<p class='jove_content'>The authors would like to acknowledge the "Ligue Nationale contre le Cancer" (Comit&#233; du Septentrion), the SIRIC ONCOLille (Grant INCa-DGOS-INSERM 6041) and the Institut pour la Recherche sur le Cancer de Lille (IRCL) for supporting this work. They would like to thank Delphine Taillieu and the animal facility staff for housing the mice and maintaining their welfare. We also thank Rapha&#235;lle Caillerez and Nathalie Jouy for their respective help in microscopy and flow cytometry.</p>

<p>动物住房和所有的实验过程由当地动物保护伦理委员会批准，CEEA.NPDC（协议no.512012），并根据实验动物的护理和使用的法国和欧洲的准则进行所有的实验。 </p><p class="jove_title"> 1. <em>在</em> C1498细胞系的<em>体外</em>表征</p><ol><li> <em>在</em> C1498细胞的<em>体外</em>培养<ol><li>制备完全RPMI（罗斯韦尔园区纪念研究所）加入50毫升胎牛血清（FBS），5 ml青霉素1640培养基（100单位/毫升）-streptomycin（100微克/毫升）中，加入500μl的50mMβ巯基乙醇的，5毫升N-2-羟乙基-N-2-乙烷磺酸（HEPES），5毫升的非必需氨基酸和5毫升丙酮酸钠至500ml RPMI培养基。 </li><li>成长C1498细胞系完全RPMI。收获在悬浮液中的细胞，通过移液，将细胞转移到50毫升管中。离心机350 XG 10分钟，然后取出superna重要的。 </li><li>在350 XG加入20毫升磷酸盐缓冲盐水（PBS）（1×）溶液，离心10分钟，并除去上清液。 </li><li>悬浮细胞在10ml荧光激活细胞分选（FACS）缓冲液（2.5克牛血清白蛋白（BSA）的粉末和2毫升在500毫升PBS溶液的0.5M乙二胺四乙酸（EDTA）溶液）。计数使用托马细胞与台盼蓝染色细胞计数后室中的细胞。 </li></ol></li><li>在C1498细胞的表型鉴定采用免疫组化和流式细胞仪分析<ol><li>细胞表面染色<ol><li>准备流式细胞仪缓冲。 </li><li>调整在FACS缓冲液中收获的细胞以10 <sup>7</sup>个细胞/ ml和分配10 <sup>6个</sup>细胞（在100μl）对每个染色实验入流式细胞管中。 </li><li>标记用100μlFACS缓冲液稀释下列抗体或它们的相关同种型对照的细胞: <ol><li>对于M前体细胞和分化细胞的arkers，标号与抗CD11b /抗CD18（1），抗-LY-6G（1），抗CD19，抗B220（2），抗NK1.1，抗细胞CD49b，抗CD4（1），抗CD8（2），抗CD3（3），抗CD21 / 35，抗CD115，抗TCRVβ抗体。 </li><li>造血干/祖细胞标记物，使用抗CD34的结合/抗CD117 /抗的Sca-1，抗CD150 /抗CD117 /抗的Sca-1，抗CD117 /抗CD127或抗独自CD16 / 32  - 生物素的抗体。 </li><li>对的细胞功能（ <em>例如</em> ，粘附，抗原呈递，共刺激分子和受体）标记物，细胞染色用抗CD18（2）/抗CD11a，抗MHC I类，抗MHC II类，抗CD31，抗CD44，抗CD80-生物素，抗CD86和抗CD274抗体。 </li></ol></li><li>孵育所有流式细胞管中流动的，在4℃下30分钟。 </li><li>洗细胞通过加入2ml FACS缓冲液中向每个管中，离心分离，在350×g离心5分钟，两次，并去除上清。 </LI&gt; <li>加入100微升的FAC缓冲液中，以每管并通过（在FACS缓冲液1/100 1/200最终稀释度）加入100微升荧光链霉亲到生物素化的缀合的抗体进行二次染色。孵育管在4℃下30分钟。 </li><li>洗细胞两次如下:在350 xg离心加2ml FACS缓冲液中向每个管中，离心管5分钟，并通过移液除去上清液。 </li><li>重悬500微升冷PBS的细胞置于冰上的细胞，用铝箔覆盖管保持他们在黑暗中。分析使用流式细胞仪<sup>10个</sup>结果。 </li></ol></li><li>细胞内染色<ol><li>通过加入125毫升4％多聚甲醛（PFA）溶液至375毫升PBS溶液制备的固定缓冲液中。 <br />注意:要在通风橱制成500毫升之4％PFA的，热400毫升PBS溶液至约60℃的搅拌板上。添加20克PFA粉末，并提高pH值直到PFA溶解。允许溶液冷却，调节pH至6.9，并补足体积至500毫升PBS中。 </li><li>通过加入0.5克皂苷和0.5g的BSA至500ml的PBS溶液制备透缓冲器。 </li><li>调整在FACS缓冲液中收获的细胞以10 <sup>7</sup>个细胞/ ml和分发10 <sup>6个</sup>细胞（100微升）为每个染色实验入流式细胞管中。离心细胞在350 XG 5分钟，去除上清。 </li><li>固定细胞在200μl的1％PFA溶液中并在4℃孵育10分钟。 </li><li>在350 XG2毫升透缓冲液添加至每个管，离心管5分钟，并通过移液除去上清液。添加100μl的透化缓冲液至每个管。 </li><li>标记用100μl下列抗体或它们相应的同种型对照的细胞透化缓冲液稀释它们后:抗CD3（2）/抗CD8（1），抗CD3（3）/一NTI-CD4（2），抗CD107b和抗CD3（3）/抗TCRVβ。 </li><li>孵育细胞在4℃下进行30分钟。 </li><li>通过加入2ml透化缓冲到每个管的两次洗细胞。离心管350 XG 5分钟，去除上清。 </li><li>添加100μl的透缓冲器向细胞。通过加入100微升荧光链霉在透化缓冲液至生物素标记的抗体稀释进行到二次染色。孵育管在4℃下30分钟。 </li><li>加2ml透化缓冲各管，在350 XG 5分钟离心管，去除上清。重复此步骤一次。 </li><li>重悬500微升冷PBS的细胞，然后将细胞在黑暗中的冰。分析用流式细胞仪<sup>10</sup>的流程的结果。 </li></ol></li></ol></li><li>细胞悬浮液在载玻片上为显微镜制备<ol><li>医管局洗10 <sup>6</sup>rvested C1498细胞（在步骤1.1.4获得）用5毫升冷的FAC缓冲液两次，稀释细胞在1ml冷的FACS缓冲液中。放置管在冰上。 </li><li>将滑入一次性室预过滤器附加卡和将这些成cytocentrifuge。 </li><li>加入100微升的FAC缓冲液中向每个腔室和过滤卡，并在4.52×g下旋转他们2分钟。 </li><li>加入100微升细胞对各室和过滤卡，和自旋细胞在4.52 xg离心2分钟。 </li><li>小心地从腔室取出幻灯片和髓过氧化物酶（步骤1.4），酯酶（步骤1.5）或月-Grünwald的吉姆萨（步骤1.6）染色之前风干的幻灯片。 </li></ol></li><li>髓过氧化物酶染色免疫<ol><li>通过在冷的甲醇中浸渍载玻片固定在载玻片细胞:丙酮（1:1）溶液，2分钟，然后空气干燥的幻灯片。 </li><li>到液体限制到含有细胞的滑动的面积，画一个词rcle周围使用防水笔细胞。 </li><li>冲洗在200μl冷PBS溶液的细胞10分钟。 </li><li>阻断细胞在200μl含有正常驴血清的10微升和10微克/毫升的纯化的抗CD16 / 32的抗体的3％BSA / PBS缓冲液中。 </li><li>应用200微升抗小鼠髓过氧化物酶（在3％BSA / PBS缓冲液稀释至20微克/毫升）的。孵育细胞O / N，在4℃的湿度室中。 </li><li>清洗细胞用200μl冷的0.1％BSA / PBS中。 </li><li>应用200微升在3％BSA / PBS缓冲液1/250稀释的抗山羊IgG抗体。孵育所述细胞在室温下在潮湿箱2小时。 </li><li>清洗细胞3次，用200μl0.1％BSA / PBS缓冲液中并两次冷PBS。 </li><li>用200μl的Hoechst的（在1微克/毫升的最终浓度）在室温2分钟在PBS中的1/1000稀释染色细胞核。 </li><li>用清水洗净，幻灯片和让他们mountin前风干G。应用安装介质1的小区中的一个下降，放置一个盖玻璃的一个边缘到幻灯片，并小心地将其放下到使用镊子将细胞。轻轻按压外盖玻璃，以消除任何气泡。 </li></ol></li><li>酯酶细胞化学<br />注:预暖的所有试剂室温。 <ol><li>固色剂的制备<ol><li>制备柠檬酸盐丙酮 - 甲醛（CAF）溶液中，添加2.5毫升柠檬酸盐溶液，6.5毫升丙酮和0.8ml 37％的甲醛至玻璃瓶中。轻轻混匀并在4℃下保存。 </li></ol></li><li>色酚AS-D氯乙酸酯酶（CAE）活性测定<ol><li>温的去离子水至37℃。 </li><li>在50ml管中，加入1毫升亚硝酸钠溶液到1ml的染料溶液。轻轻混匀，静置2分钟。加40毫升预热的去离子水，5毫升的pH 6.3缓冲液浓缩物和1的萘酚AS-D氯乙酸溶液。混合并转移到一个罐子科普林。 </li><li>固定细胞到幻灯片（见1.3节），用于与CAF溶液（见步骤1.5.1.1）30秒，并洗玻片45秒，用去离子水。 </li><li>转移滑入这是在步骤1.5.2.2中制备的溶液，并在37°C孵育载玻片在避光湿度室30分钟。 </li><li>干燥载玻片，然后通过浸入冲洗他们在去离子水中2分钟。 </li><li>通过加入苏木溶液几滴孵育他们1分钟染液细胞。 </li><li>与中性水（pH 7）洗涤载玻片并允许风干。应用安装介质2向小区中的一个下降，放置一个盖玻璃的一个边缘到滑动并小心地将其放下到使用镊子将细胞。轻轻按压外盖玻璃，以消除任何气泡。 </li></ol></li><li>阿尔法萘丁酯酶（NBE）活性测定<ol><li>使用前暖α萘丁解到37℃。 </li><li> NIT稀释钠一片RITE 6.25毫升去离子水。 </li><li>在50ml管中，加入为1.5ml亚硝酸钠片剂溶液和1.5毫升副玫瑰苯胺溶液。轻轻混合，并允许溶液静置5分钟。补充用40ml磷酸盐缓冲溶液中的溶液。通过仔细加入10 N滴加氢氧化钠带给pH值为6。加入5毫升α萘丁解决方案，混合整个解决方案，并将其转移到一个罐子科普林。 </li><li>用在RT下CAF溶液固定细胞在载片上，持续10秒，冲洗45秒，用去离子水。 </li><li>转印滑入包含在步骤1.5.3.2制备并一起1小时，在37℃的湿度室中孵育，同时避光溶液中的科普林缸。 </li><li>冲洗的幻灯片在中性水中2分钟（pH值为7）和空气干燥。 </li><li>染液与亚甲基蓝溶液中的细胞在幻灯片上加入几滴孵育4分钟。 </li><li>浸入deio幻灯片的发布水2分钟并允许风干。挂载幻灯片，应用安装介质2向小区中的一个下降，放置在盖玻璃的一个边缘到幻灯片，并小心地将其放下到使用镊子将细胞。轻轻按压外盖玻璃，以消除任何气泡。 </li></ol></li></ol></li><li>五月 - 格伦沃尔德姬姆萨（MGG）染色<ol><li>通过幻灯片浸入含五月 - 格伦沃尔德溶液中3分钟的科普林氏缸染色细胞（以1.3节准备）。 </li><li>转移的幻灯片到含有1分钟的pH 6.8的缓冲溶液中的科普林缸。 </li><li>将它们放置在含有10分钟的吉姆萨R染料溶液（在pH 6.8的缓冲液稀释到1/20）一个科普林缸染色的幻灯片。洗与中性水（pH 7）的幻灯片，持续10秒。 </li><li>漏和风干的幻灯片。通过施加固定介质2到细胞上的一滴装入滑动。将盖板玻璃的一个边缘到幻灯片，并小心地放下它到细胞中使用镊子。轻轻按压外盖玻璃，以消除任何气泡。 </li></ol></li></ol><p class="jove_title"> 2. <em>在体内</em>发育和急性白血病的表征</p><p>注:4周大的雌性同类系的C57BL / 6J-Ly5.1小鼠无特定病原体的条件下维持（ <em>即，</em>在无菌环境中）。当他们周5和6岁之间的小鼠注射。 </p><ol><li>静脉注射C1498细胞<ol><li>吹打收获悬浮培养细胞C1498。转移细胞到一个50毫升管中并离心，在350×g离心10分钟。清洗细胞在10ml冷PBS两次，并准备在PBS中的10 <sup>7个</sup>细胞/ ml的细胞悬浮液。广场上冰的细胞悬浮液进行注射前。 </li><li>鼠标放置在一个限制器和一个层流罩进行无菌条件下注入。 </li><li>使用29G针，用注射器注入细胞进入尾静脉。把握tail在远端，并用70％的乙醇中浸泡过的纱布海绵消毒它。检查，以确保有在注射器没有气泡，然后缓缓注入100微升C1498细胞悬浮液（10 <sup>6个</sup>细胞）至尾静脉。 </li><li>注射后，从尾部取出针，并通过施加压力以在注射部位中的无菌纱布海绵控制任何出血。返回动物笼子里，并仔细检查其健康在接下来的几个小时和几天。 </li></ol></li><li>眼眶采血<ol><li>监视PBS-和C1498-注射的小鼠的为白血病病（ <em>例如</em> ，立毛，从组隔离，并减少或在笼不运动）的迹象的行为。 <br />注意:这通常发生17至19天之间的细胞注射后。 </li><li>只是安乐死之前执行眼眶采血（见步骤2.2.7）在无菌条件下在层流引擎盖下加热灯，以防止体温过低。 </li><li>麻醉，用氯胺酮150 mg / kg和赛拉嗪在10毫克/公斤。通过稀释1.5毫升氯胺酮和0.5ml甲苯噻嗪的18毫升PBS溶液制备麻醉剂溶液。 </li><li>麻醉控制和白血病的小鼠。用200微升每10g使用26G针小鼠的麻醉剂溶液的腹膜内注射和1ml注射器进行。检查踏板反射消失，确认麻醉。 </li><li>插入毛细管进入眼睛的内眦。血液将上升从眼窝窦入毛细管。通过轻轻地用无菌纱布海绵施加压力到眼睛控制出血。 <br />注:100〜200微升血液的体积可以使用这种技术来收集。 </li><li>收集血液到EDTA管中，并分离单核细胞前在冰上样品存储。 </li><li>使用颈椎脱位安乐死鼠标和进行隔离器官（第2.3节）。 </li></ol></li><li>器官和细胞分离<ol><li>机关隔离<ol><li>放置安乐死鼠标到其上的塑料板回用针引脚动物的脚，以促进器官隔离。消毒用70％的乙醇进行的切口之前鼠标。 </li><li>使用无菌剪刀，从腹部皮肤到颈部执行腹侧切开。切断通过腹壁来访问肝脏。通过胸廓和访问肺部隔膜切。移动至肠的侧面和除去使用无菌剪刀和镊子脾脏。 </li><li>以分离骨髓，切腿在使用无菌剪刀接头上方的股骨的顶部。通过轻轻拉动断开股骨胫骨，然后取出使用镊子和剪刀骨头的皮肤和肌肉。 </li><li>放置在含有冷PBS 50ml管各器官和骨骼，并将它们放置在冰上。 </li></ol></li><li>从器官细胞的分离<ol><li>破坏细胞前称量脾脏。通过在一个50ml管中使用的针筒柱塞按压它们通过一个70微米的过滤器机械地扰乱脾，肺和肝脏，并收集细胞为30个毫升冷PBS中。 </li><li>收集的骨髓细胞，将股骨和胫骨在培养皿中在冰上，使用无菌剪刀剪四肢，并通过插入附连到含有5毫升冷的PBS 10ml的注射器26G针冲出骨髓。 <ol><li>通过将细胞悬浮液通过针/注射器破坏骨髓细胞，并通过一个70微米的过滤器，在50毫升管过滤细胞悬浮液。 </li></ol></li><li>离心机所有含每种器官和骨髓细胞在350×g离心10分钟的管子。弃去上清液，并重新悬浮在2毫升的裂解缓冲液（1x）和细胞从肺和骨髓收集细胞轻轻上下吹打该混合物从肝脏和脾脏s隔离在5ml裂解缓冲液（1×）的。管填充50毫升冷PBS。 </li><li>离心细胞，在350×g离心10分钟。重悬在FACS缓冲液将细胞进行流式细胞术分析或制备细胞显微术。算使用托马细胞计数室中的细胞用台盼蓝染色它们之后。 </li></ol></li></ol></li><li>从机关分离出来流式细胞仪分析细胞的细胞表面染色<ol><li>在流式细胞术管的流动，标签从器官中分离用10μg/ ml，在100微升的FAC缓冲液中纯化的抗CD16 / 32抗体的有10 <sup>6个</sup>细胞。 </li><li>至10 <sup>6个</sup>骨髓细胞，添加在FACS缓冲液稀释抗体的下列抗体或组合以及它们相应的同种型对照的100微升:抗CD11b /抗CD3（1）/抗的Ly6C /抗Ly6G（2），抗B220（1）/anti-CD45.2/anti-CD19，抗CD115 /抗CD3（1）/一个TI-的Ly6C /抗Ly6G（2），抗CD45.2，抗Ly6G（2），抗CD11b，抗CD115或单独为补偿设定抗CD19。 </li><li>到的脾细胞中，添加以下抗体的100μl的和其相应的同种型对照在FACS缓冲液稀释:（1抗CD11b /抗CD3（1）/抗的Ly6C /抗Ly6G（2），抗B220的组合）/anti-CD45.2/anti-CD19，抗CD45.2，抗Ly6G（2），抗CD11b或抗CD19补偿设置。 </li><li>肺和肝细胞，添加抗CD45.2抗体的100微升，并在FACS缓冲液1/100稀释其相应的同型对照。 </li><li>孵育所有30分钟的细胞溶液在4℃下。 </li><li>通过加入2ml FACS缓冲液中向每个管洗涤细胞。离心管在350 xg离心5分钟，并通过移液弃去上清。重复此步骤一次。 </li><li>重悬标记细胞在500微升冷PBS。置于冰上的电池和交流电进行术前流避光quisition和分析<sup>10。</sup> </li></ol></li><li>外周血单个核细胞和免疫荧光染色的隔离流式细胞分析<ol><li>启动协议之前，预温分离溶液至室温。 </li><li>转移血样（100至200微升来自步骤2.2获得）到微量离心管中，并加入PBS / 1mM EDTA的溶液，直至溶液体积为500微升。仔细层500微升含有使用30G针和1ml注射器血液中的溶液下分离溶液。不要混合血液和分离解决方案。 </li><li>在800 XG（无制动器），在室温20 min离心管。离心后，收集用吸管蜂窝环（不透明的白色层）。转移细胞到一个离心管中。 <br />注:不透明白色层含有淋巴细胞以及单核细胞和下层之间出现 - 分离溶液 - 和上层。 </li><li>加入1ml的PBS溶液，并离心管中，在350×g离心10分钟。悬浮细胞在600微升FACS缓冲液中。 </li><li>添加10μg/ ml的纯化的抗CD16 / 32的抗体与100微升的细胞悬液分配到六个独立的管（每100微升）。 </li><li>标记的细胞用100μlFACS缓冲液稀释下列抗体或它们的相关同种型对照的:抗CD3（1）/抗B220（1）/anti-CD45.2和抗的Ly6C /抗CD115的结合/anti-CD45.2或抗CD45.2，独自补偿设置抗CD115。 </li><li>孵育所有试管在4℃下30分钟。 </li><li>通过加入2ml FACS缓冲液中向每个管洗涤细胞，然后在350 xg离心离心管10分钟，并使用移液管弃上清。 </li><li>重悬标记细胞在500微升冷PBS。置于冰上的细胞和流式细胞仪采集和分析<sup>10</sup>执行流之前，避光。 </li></ol></li><li>骨髓细胞悬液在玻片上显微镜准备<ol><li>按照1.3节所述的步骤，但在步骤1.3.1，使用10 <sup>5</sup>骨髓细胞，在步骤1.3.4，旋细胞到每个室在72.26 XG 10分钟。 </li></ol></li><li>利用骨髓细胞酯酶活性测定<ol><li>要进行骨髓细胞化学酯酶试验，从出发步骤1.5至1.5.3.7。 </li></ol></li><li>骨髓细胞的五月-Grünwald的吉姆萨染色<ol><li>染色骨髓细胞，按照1.6节所述的协议，但在步骤1.6.1，孵化在5  - 格伦瓦尔德解决方案的幻灯片5分钟。 </li></ol></li></ol>

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</ol>

<p class='jove_content'>The authors declare that they have no competing financial interests.</p>

<p class="jove_content">在以前的研究中，C1498细胞系被描述为急性粒<sup><sup>5，6</sup></sup>粒单核细胞或NKT <sup>7</sup>细胞白血病的诱导。然而，在文献中示范的数据是不存在或不完整的。这里介绍的协议使用不同的技术，如流式细胞术，免疫荧光，MGG染色和细胞化学测定法，以表征培养的C1498细胞，并确定在小鼠中诱导它们被注射后的白血病的性质。 </p><p class="jove_content">当我们<em>在体外</em>培养的C1498细胞表型免疫染色后和流式细胞术进行分析，我们观察到一些限制，因为这些细胞中表达已在文献<sup>6,7</sup>之前已经描述几个细胞表面造血标记。与我们的结果一致，拉贝勒<em>等人</em>没有使用流动cytomet观察C1498细胞成熟TCR的细胞表面表达RY染色。然而，他们认为他们是NKT细胞系，他们发现CD3ε和TCRVβ8.2的mRNA <sup>7</sup>之后。我们还观察到TCRVβ链和CD3ε分子的细胞内表达在大多数细胞（&gt; 70％），但无法确定他们的造血谱系因为也有在Mac-3分子的伴随的细胞内表达。 </p><p class="jove_content">髓过氧化物酶，MGG染色和评估分析使用细胞化学功能酯酶表明，C1498细胞系具有髓系起源和组成monoblasts和原粒细胞。这些结果是一致的，在流量获得染色流式细胞仪分析认为Mac-3 <sup>+</sup>细胞的百分比。虽然不是定量的，这些步骤代表键实验中进行。事实上，它们仍然存在，到目前为止，用于表征表达没有或f造血细胞的谱系和分化阶段最好现有的方法EW特定表型标记。 </p><p class="jove_content">流式细胞仪染色用于演示急性白血病的小鼠同类后发展C1498细胞静脉注射有帮助的。的CD45.2 <sup>+</sup> C1498细胞渗入外周血和各种器官进行分离，并测定它们的频率。还进行免疫定量分析后，分析内在髓质和脾细胞。当器官被检查的C1498细胞表型，因为它们表达了几个造血标记中遇到的限制（只有少数为B220 <sup>+）。</sup>来定义观察急性白血病的性质，可能 - Grünwald的Giemsa染色和单核细胞的活动进行分析并使用骨髓进行粒酯酶。结果表明，C1498细胞保留其髓和单核形态和功能，揭示单核细胞白血病的发病。 </p><p clasS ="jove_content"&gt;在考虑在本协议中所描述的关键步骤，应特别注意进行细胞化学反应和MGG染色时，由于pH值的错误可能会导致结果不正确的解释给予pH值。例如，α萘丁酸酯酶活性只在pH 6.0是特定于单核细胞，因为粒细胞和淋巴细胞也可以染色阳性此测试在较高的pH值。凝视细胞进行染色MGG之前不建议的，我们发现只有CAF固定使用C1498细胞进行酯酶当细胞化学反应提供了令人满意的结果。为了保持CD115分子的表达，并通过流式细胞其检测仪中，所有的样品（ <em>例如</em> ，血液，骨髓和脾细胞）应在手术过程中保持在冰上。如果没有染色流式细胞仪或/和免疫荧光实验，抗体的参考，其存储重新观察表彰和其稀释液应进行检查。在材料/设备表中指定的引用已经被选择用于流式细胞术或免疫荧光的应用程序。初级/次级抗体或它们的共轭荧光团可能已经失去了他们的活动，由于不恰当的存储（ <em>例如</em> ，暴露于光或热），稀释不当，大量的冷冻/解冻或使用受污染的缓冲区。运行阳性对照，以确保它们正常工作。使用小鼠骨髓或已知为表达目的蛋白脾来源的细胞。为了避免高的背景和非特异性染色，确保将细胞洗净，并保持在高湿度（对于免疫荧光），并且作为指示抗体稀释。使用相同的浓缩和稀释的同种型对照抗体和第一抗体精确地确定样品中的背景水平。酯酶细胞化学实验，试剂可通过使用含有纯化的小鼠脾粒（Ly6G <sup>+）</sup>和单核细胞（CD115 <sup>+）</sup>细胞阳性和阴性对照载玻片进行测试。 </p><p class="jove_content">在这项研究中所描述的过程表明，许多的在小鼠中观察到C1498细胞注射后的白血病功能共享与人急性单核细胞白血病<sup>11,12</sup>共同特点。侵入白血病细胞导致减少成熟和未成熟（祖细胞和前体）骨髓造血细胞。 C1498细胞存在于外周血中的高频率（&gt; 20％），因为是单核细胞。观察肝脾肿大从白血病细胞的浸润导致，并在B淋巴细胞和骨髓细胞显著上升也观察陪脾肿大。当血液中的血小板数均使用血液分析仪血小板减少，估计也观察到。 </p><p class="jove_content">这是秀N， <em>使用体外</em>实验，即C1498细胞通过分泌可溶性因子<sup>13</sup>抑制正常小鼠造血功能。在几个肿瘤小鼠模型中，未成熟的骨髓细胞（包括单核细胞和粒细胞）也已经显示，从骨髓迁移到脾，在那里它们抑制抗肿瘤特异性T细胞活化和增殖<sup>14。</sup>因而，在骨髓中观察到，在造血细胞中的减少可能导致从任造血和/或从他们的移民缺乏。这后一种机理可以解释单核细胞增多的外周血中存在或放大的骨髓级分在脾脏的观察。还可以想到这些细胞可能已被从改进的脾造血而得。事实上，稳态条件下，脾B细胞的部分子集被确定为成熟的B淋巴细胞前体<sup>15。</sup>此外，炎症的条件下，配有ullary干细胞和祖细胞已被证明搬迁到脾诱导产生成熟单核细胞<sup>16。</sup>这个协议不允许我们得出关于所涉及的白血病的发展，和附加的功能以及分子检测应采用这样做机制结论。然而，这些数据包括有关急性单核细胞白血病的临床特点的详细信息，并帮助研究人员评估和了解新的治疗药物的影响。 </p>

<p class="jove_content">急性髓性白血病（AML）的特征在于被阻止在成熟的不同阶段的造血骨髓细胞的不受控制的增殖。这种失调可影响粒，单核细胞，红细胞或megaryocytic分化途径<sup>1。</sup> AML细胞积聚在骨髓，导致受损的造血，这导致血小板减少，淋巴细胞和贫血。白血病细胞也侵入血液和非淋巴器官。 </p><p class="jove_content">的C1498小鼠模型已使用了几十年作为急性白血病，因为癌细胞从白血病10月龄的C57BL / 6（H-2 <sup>B）</sup>雌性小鼠在1941年分离出一个模型的文献描述了侵入血液，造血器官（ <em>例如</em> ，脾和淋巴结）和非造血器官（ <em>例如</em> ，肝，肺，卵巢，和肾）由高度增殖C1498细胞后，他们通过在注入travenous，皮下或腹膜内途径进入易感小鼠<sup>2-4。</sup>然而，这种小鼠模型报道诱发要么粒<sup>2,5</sup>或<sup>6</sup>粒单核细胞白血病。最近，发表在2002年的一项研究说明这种类型的癌症如鼠NKT细胞白血病<sup>7。</sup>因此，该文献的不同对本C1498细胞系的性质和它在小鼠中诱导的相关性白血病。这些差异主要是由于缺乏详细的和有关的细胞更新的公布的信息，并在一般的白血病的疾病，因为许多研究是在1950年进行的 -  70年代。 </p><p class="jove_content">在这里，我们提供了一个详细的协议来描述如何表征C1498细胞，并分析该被诱导可以通过静脉注射到小鼠白血病病的性质。这个协议的第一部分是专门为那些<em>在体外</em>培养的细胞C1498的描述。荧光抗针对表面和细胞内的造血标记的尸体被用于使用流式细胞仪，以确定其表型。髓过氧化物酶的存在下，用免疫荧光显微镜进行评估，其造血谱系和分化阶段用细胞化学以评估酯酶的活性进行评价，并进行可能 - Grünwald的吉姆萨染色。然后C1498细胞注射到小鼠中，而且诱导的急性白血病病在本手稿的第二部分进行说明。相同的技术被用于确定频率和白血病和固有细胞在骨髓，外周血，脾和非造血器官（肝和肺）的表型。 </p><p class="jove_content">此协议是高度可重复的，这里提供的数据将有助于科研人员评估新的治疗策略的影响。此白血病小鼠模型已被用于测试免疫接近ND不同肿瘤化疗药物<sup>8,9。</sup>其功效通过测定肿瘤负荷和存活率的演变进行评价。该协议可以被用来提供关于白血病和其它造血细胞群的治疗过程中的分布和生活的其他信息。 </p>

<table><tbody><tr><td>C1498 cell line</td><td>ATCC</td><td>TIB 49</td><td></td></tr><tr><td>C57BL/6J-Ly5.1</td><td>Charles River</td><td>B6.SJL-Ptprc a Pep3 b/BoyCrl</td><td></td></tr><tr><td>&lt;strong&gt;Cells culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Mercaptoethanol, Gibco</td><td>ThermoFisher Scientific</td><td>21985</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher Scientific</td><td>10270</td><td></td></tr><tr><td>HEPES, Gibco (1 M)</td><td>ThermoFisher Scientific</td><td>15630</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution, Gibco&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11140</td><td></td></tr><tr><td>Penicillin-Streptomycin, Gibco&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15140</td><td></td></tr><tr><td>RPMI 1640 Medium (Gibco, GlutaMAX Supplement)</td><td>ThermoFisher Scientific</td><td>61870</td><td></td></tr><tr><td>Sodium Pyruvate, Gibco (100 mM)</td><td>ThermoFisher Scientific</td><td>11360</td><td></td></tr><tr><td>&lt;strong&gt;Flow cytometry staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse B220 APC (1)</td><td>ebiosciences</td><td>17-0452</td><td>clone RA3-6B2, final dilution 1/100</td></tr><tr><td>anti-mouse B220 biotin (2)</td><td>ebiosciences</td><td>13-0452</td><td>clone RA3-6B2, 1/400</td></tr><tr><td>anti-mouse CD3 eFluor450 (1)</td><td>ebiosciences</td><td>48-0032</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE (2)</td><td>BD biosciences</td><td>555275</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE-Cy5 (3)</td><td>ebiosciences</td><td>15-0031</td><td>clone 145-2C11, 1/100</td></tr><tr><td>anti-mouse CD4 APC (1)</td><td>ebiosciences</td><td>17-0041</td><td>clone GK1.5, 1/500</td></tr><tr><td>anti-mouse CD4 PE (2)</td><td>ebiosciences</td><td>12-0041</td><td>clone GK1.5, 1/200</td></tr><tr><td>anti-mouse CD8 biotin (1)</td><td>ebiosciences</td><td>13-0081</td><td>clone 53-6.7, 1/100</td></tr><tr><td>anti-mouse CD8 eFluor450 (2)</td><td>ebiosciences</td><td>48-0081</td><td>clone 53-6.7, 1/500</td></tr><tr><td>anti-mouse CD11a biotin</td><td>ebiosciences</td><td>13-0111</td><td>clone M17/4, 1/100</td></tr><tr><td>anti-mouse CD11b PE&amp;nbsp;</td><td>ebiosciences</td><td>12-0112</td><td>clone M1/70, 1/200</td></tr><tr><td>anti-mouse CD16/32 biotin</td><td>ebiosciences</td><td>13-0161</td><td>clone 93, 1/400</td></tr><tr><td>purified anti-mouse CD16/32 (FcR blocking)&amp;nbsp;</td><td>BD biosciences</td><td>553141</td><td>clone 2.4G2</td></tr><tr><td>anti-mouse CD18 biotin (1)</td><td>ebiosciences</td><td>13-0181</td><td>clone M18/2, 1/100</td></tr><tr><td>anti-mouse CD18 FITC (2)</td><td>ebiosciences</td><td>11-0181</td><td>clone M18/2, 1/50</td></tr><tr><td>anti-mouse CD19 PE</td><td>ebiosciences</td><td>12-0193</td><td>clone 1D3, 1/200</td></tr><tr><td>anti-mouse CD21/35 PE</td><td>ebiosciences</td><td>12-0211</td><td>clone 8D9, 1/50</td></tr><tr><td>anti-mouse CD31 PE</td><td>ebiosciences</td><td>12-0311</td><td>clone 390, 1/100</td></tr><tr><td>anti-mouse CD34 eFluor660</td><td>ebiosciences</td><td>50-0341</td><td>clone RAM34, 1/20</td></tr><tr><td>anti-mouse CD44 PE</td><td>BD biosciences</td><td>553134</td><td>clone IM7, 1/50</td></tr><tr><td>anti-mouse CD45.2 FITC&amp;nbsp;</td><td>ebiosciences</td><td>11-0454</td><td>clone 104, 1/100</td></tr><tr><td>anti-mouse CD49b/Pan NK PE</td><td>&amp;nbsp;BD biosciences</td><td>553858</td><td>clone DX5, 1/50</td></tr><tr><td>anti-mouse CD80 biotin</td><td>ebiosciences</td><td>13-0801</td><td>clone 16-10A1, 1/200</td></tr><tr><td>anti-mouse CD86 biotin</td><td>ebiosciences</td><td>13-0862</td><td>clone GL1, 1/200</td></tr><tr><td>anti-mouse CD107b (Mac-3) PE</td><td>ebiosciences</td><td>12-5989</td><td>clone M3/84, 1/40</td></tr><tr><td>anti-mouse CD115 PE</td><td>ebiosciences</td><td>12-1152</td><td>clone AFS98, 1/100</td></tr><tr><td>anti-mouse CD117 eFluor450</td><td>ebiosciences</td><td>48-1171</td><td>clone 2B8, 1/100</td></tr><tr><td>anti-mouse CD127 PE</td><td>ebiosciences</td><td>12-1271</td><td>clone A7R34, 1/100</td></tr><tr><td>anti-mouse CD150 APC</td><td>ebiosciences</td><td>17-1501</td><td>clone 9D1, 1/20</td></tr><tr><td>anti-mouse CD274 (PD-L1) biotin</td><td>ebiosciences</td><td>13-5982</td><td>clone MIH5, 1/200</td></tr><tr><td>anti-mouse Ly-6A/E (Sca-1) PE</td><td>ebiosciences</td><td>12-5981</td><td>clone D7, 1/100</td></tr><tr><td>anti-mouse Ly-6C APC</td><td>ebiosciences</td><td>17-5932</td><td>clone HK1.4, 1/200</td></tr><tr><td>anti-mouse Ly-6G&amp;nbsp; (Gr-1) biotin (1)</td><td>ebiosciences</td><td>13-5931</td><td>clone RB6-8C5, 1/400</td></tr><tr><td>anti-mouse Ly-6G FITC (2)</td><td>ebiosciences</td><td>11-9668</td><td>clone 1A8, 1/50</td></tr><tr><td>anti-mouse MHC class I (H-2Db) biotin</td><td>ebiosciences</td><td>13-5999</td><td>clone 28-14-8, 1/50</td></tr><tr><td>anti-mouse MHC class II (I-A/I-E) PE-Cy5</td><td>ebiosciences</td><td>15-5321</td><td>clone M5/114.15.2, 1/1000</td></tr><tr><td>anti-mouse NK1.1 PE</td><td>BD biosciences</td><td>557391</td><td>clone PK136, 1/50</td></tr><tr><td>anti-mouse TCRVb FITC</td><td>BD biosciences</td><td>553170</td><td>clone H57-597, 1/50</td></tr><tr><td>streptavidin PE-Cy5</td><td>ebiosciences</td><td>15-4317</td><td>&amp;nbsp;&amp;nbsp; 1/200</td></tr><tr><td>Immunofluorescence staining reagents</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse myeloperoxidase (heavy chain antibody)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>sc-16129</td><td>1/10 (20 &amp;micro;g/ml)</td></tr><tr><td>&amp;nbsp;anti-goat IgG (Texas Red coupled antibody)</td><td>Jackson Immunoresearch</td><td>705-076-147</td><td>1/250</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immunoresearch</td><td>017-000-001</td><td></td></tr><tr><td>Hoechst solution</td><td>BD Biosciences</td><td>561908</td><td>1/1000</td></tr><tr><td>Mounting medium 1 (Fluoromount)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4680</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>&lt;strong&gt;Esterase cytochemical staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Naphtol AS-D chloroacetate solution</td><td>Sigma-Aldrich</td><td>911</td><td></td></tr><tr><td>Dye solution (Fast Red Violet LB Base solution)</td><td>Sigma-Aldrich</td><td>912</td><td></td></tr><tr><td>pH6.3 buffer concentrate (TRIZMAL)</td><td>Sigma-Aldrich</td><td>913</td><td></td></tr><tr><td>Sodium nitrite solution</td><td>Sigma-Aldrich</td><td>914</td><td></td></tr><tr><td>Citrate solution</td><td>Sigma-Aldrich</td><td>915</td><td></td></tr><tr><td>Hematoxylin solution</td><td>Sigma-Aldrich</td><td>GHS3</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Formaldehyde solution, 37%</td><td>Sigma-Aldrich</td><td>F1635</td><td></td></tr><tr><td>&amp;alpha;-naphthyl butyrate solution</td><td>Sigma-Aldrich</td><td>1801</td><td></td></tr><tr><td>Phosphate buffer solution</td><td>Sigma-Aldrich</td><td>1805</td><td></td></tr><tr><td>Sodium nitrite Tablet</td><td>Sigma-Aldrich</td><td>1809</td><td></td></tr><tr><td>Pararosaniline solution</td><td>Sigma-Aldrich</td><td>1804</td><td></td></tr><tr><td>Methylene blue solution</td><td>Sigma-Aldrich</td><td>1808</td><td>1/10</td></tr><tr><td>mounting medium 2 (Clearmount)</td><td>Invitrogen</td><td>00-8010</td><td></td></tr><tr><td>&lt;strong&gt;May-Gr&amp;uuml;nwald Giemsa staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>May-Gr&amp;uuml;nwald solution</td><td>RAL Diagnostics</td><td>320070</td><td></td></tr><tr><td>Giemsa R solution&amp;nbsp;</td><td>RAL Diagnostics</td><td>320310</td><td>&amp;nbsp;&amp;nbsp; 1/20</td></tr><tr><td>pH 6.8 buffer solution</td><td>RAL Diagnostics</td><td>330368</td><td></td></tr><tr><td>&lt;strong&gt;Others materials, reagents and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketamine 1000 (100 mg/ml)</td><td>VIRBAC</td><td>3597132111010</td><td></td></tr><tr><td>Xylazine SEDAXYLAN (20 mg/ml)</td><td>CEVA</td><td></td><td></td></tr><tr><td>Bovin albumin serum (BSA) powder</td><td>ThermoFisher Scientific</td><td>BP671</td><td></td></tr><tr><td>PBS solution (1x concentrate)</td><td>ThermoFisher Scientific</td><td>14190</td><td></td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Ultra Pure 0.5 M EDTA solution, pH 8.0</td><td>ThermoFisher Scientific</td><td>15575</td><td></td></tr><tr><td>Separating solution (Pancoll Mouse)</td><td>PANBIOTECH</td><td>P04-64100</td><td></td></tr><tr><td>Lysis buffer (Red Blood Cells Lysing Buffer) (10x)</td><td>BD Biosciences</td><td>555899</td><td>1/10</td></tr><tr><td>NaOH 10 N</td><td>ThermoFisher Scientific</td><td>SS267</td><td></td></tr><tr><td>Trypan blue solution (0.4 %)</td><td>ThermoFisher Scientific</td><td>15250-061</td><td>1/2</td></tr><tr><td>50 ml tube (Falcon)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer (Falcon)&amp;nbsp;</td><td>Corning Life Sciences</td><td>352350</td><td></td></tr><tr><td>&amp;nbsp;Chamber &amp;amp; filter card (EZ Cytofunnel Shandon)</td><td>Thermo Scientific</td><td>A78710003</td><td></td></tr><tr><td>Microscope Cover Glasses, 24 x 24mm</td><td>Knittel Glass</td><td>VD1 2424 Y100</td><td></td></tr><tr><td>Slides (Starfrost - ground edges 90)</td><td>Knittel Glass</td><td>VS1137# 077FKB</td><td></td></tr><tr><td>EDTA Tube</td><td>Greiner Bio-One</td><td>454034</td><td></td></tr><tr><td>Pasteur Pipette 150 mm (capillary tube)</td><td>Fisherbrand</td><td>1154-6963</td><td></td></tr><tr><td>26 G needle</td><td>Terumo</td><td>NN-2613R</td><td></td></tr><tr><td>&amp;nbsp;insulin syringe and needle 29 G</td><td>Terumo</td><td>BS05M2913</td><td></td></tr><tr><td>30 G needle&amp;nbsp;</td><td>Becton &amp;amp; Dickinson</td><td>304000</td><td></td></tr><tr><td>Flow cytometry tubes (blue)</td><td>Beckman Coulter</td><td>2523749</td><td></td></tr><tr><td>Water-repellent pen (Dakopen)</td><td>Dako</td><td>S200230</td><td></td></tr><tr><td>Sharp sterile scissors&amp;nbsp;</td><td>Nessi-care</td><td>SCI-01</td><td></td></tr><tr><td>Sterile forceps</td><td>Dominique Dutscher</td><td>956506</td><td></td></tr><tr><td>Thoma cell counting chamber</td><td>VWR</td><td>631-0397</td><td></td></tr><tr><td>Petri Dishes (Fisherbrand Plastic)</td><td>ThermoFisher Scientific</td><td>S33580A</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 ml)</td><td>ThermoFisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Cylindrical Restrainer 15 - 30 gm</td><td>Stoelting</td><td>51338</td><td></td></tr><tr><td>Shandon Cytospin 3 Cytocentrifuge</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SS-10L</td><td></td></tr><tr><td>1 ml syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.08543</td><td></td></tr><tr><td>Sterile gauze sponges</td><td>URGO</td><td>501580</td><td></td></tr></tbody></table>

a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model

<p class="jove_content">已经执行了静脉注射C1498细胞进同源或同类系小鼠自1941年这些注射导致急性白血病的发展。然而，这种疾病的性质并没有得到很好的文献记载。这里，我们<em>在体外</em>表征C1498细胞和用于确定<em>在体内</em>诱发白血病的性质提供了技术的协议。这个过程的第一部分聚焦在确定所述造血谱系和培养C1498细胞分化的阶段。为了实现这一点，多参数流式细胞染色用于检测造血细胞标记。免疫荧光显微镜，细胞化学和五月-Grünwald的吉姆萨染色，然后进行评估髓过氧化物酶的表达，酯酶和细胞形态的活性，分别。该协议的第二部分专门描述了<em>在</em>诱导白血病病体内。后者可通过测定血液中的白血病和固有细胞的频率来实现的，使用特定的染色和流式细胞术造血器官（ <em>例如</em> ，骨髓和脾）和非淋巴组织（ <em>例如</em> ，肝，肺）分析。白血病的性质然后使用月-Grünwald的吉姆萨染色和染色为在骨髓中具体酯酶证实。这里，我们提出使用在年龄匹配C1498-和PBS注射的小鼠中这个协议得到的结果。 </p>

<p class="jove_content">表征C1498小鼠模型中，我们进行了两个主要步骤。首先，将C1498细胞进行表征，以确定<em>其体外</em>造血谱系和成熟阶段（ <strong>图1）。</strong>然后将这些细胞注射入同类小鼠和诱导白血病疾病的性质进行了评估，以确定不同的功能:白血病细胞浸润，它们的表型，所述造血细胞的骨髓一个量化（成熟和祖细胞/前体），该频率的C1498细胞和血液中的成熟的造血细胞和器官肿胀的评价（在脾，肝和肺）和细胞组合物。 </p><p class="jove_content">为了表征<em>体外</em> C1498细胞表型，细胞标记与针对分子的抗体是由造血前体和成熟细胞（ <strong>表1）</strong>表示，并且使用流量cytomet结果进行分析RY。在C1498细胞呈阳性的Mac-1（CD11b的/ CD18）（〜7％），B220（&gt; 25％）的细胞表面表达，它们显示CD3ε，T细胞受体（TCR）Vβ链和Mac的胞内表达-3（ <strong>图2A和B）。</strong>细胞为阴性的细胞表面标记物Ly6G，的Ly6C，CD115，CD21 / CD35，CD19，CD3，CD4，CD8，NK1.1，和泛NK细胞分子和对于CD4和CD8的胞内表达（数据未示出） 。然后，他们被检查的造血干细胞和祖细胞（ <strong>表1）</strong>的标志物。他们也为阴性CD117，CD34，的Sca-1，CD150和CD16 / 32的细胞表面表达（数据未显示）。然后将这些白血病细胞进行试验，以确定粘附，抗原呈递和协同刺激分子的表达。细胞中表达的表面标记的LFA-1（CD11a / CD18），CD44，CD31（PECAM-1），和H-2D <sup>b</sup>和为阴性MHC II类，CD80，CD86和CD274（数据未显示）。 C1498细胞ŧherefore表示既髓（MAC-1，Mac的-3）和淋巴标记（B220，CD3，TCR）。 </p><p class="jove_content">为了更好地表征自己的造血系统，髓过氧化物酶的表达，用免疫荧光显微镜进行评估。所有的细胞呈阳性的髓过氧化物酶，验证他们的骨髓来源（ <strong>图3A）。</strong>大多数细胞还沾着积极为α萘丁酯酶（ <strong>图3B，左图</strong> ），其中一些染色的色酚AS-D氯乙酸酯酶（黑色箭头）（ <strong>图3B，右图</strong> ）。结果表明，该细胞含有单核细胞和粒细胞的混合物。进行五月-Grünwald的吉姆萨染色后，观察到C1498细胞具有高核质比，以显示一个爆炸样形态，在细胞核中3到5的核仁，核周卤素，大量液泡和嗜碱性细胞质（ <strong>图3C</strong> ）。钍美国，C1498细胞系是由monoblasts和原粒细胞。 </p><p class="jove_content">的C1498细胞（CD45.2 <sup>+）</sup>然后静脉注射到CD45.1 <sup>+</sup>小鼠。小鼠屈服于所述细胞注射后17至19天。这些小鼠处死，使他们死于这种疾病在他们的白血病类型可以进行分析。对照小鼠，该注射用PBS，在用于比较的相同的时间点进行分析。在C1498细胞注射的小鼠显示C1498细胞大量浸润到他们的骨髓，这表现在细胞的爆炸状外观后五月-Grünwald的吉姆萨染色进行（ <strong>图4A）。</strong>他们还保存他们的单核细胞和粒表型（ <strong>图4B和C），</strong>显示出单核和髓细胞的积累是急性单核细胞白血病的特征。 </p><p class="jove_content" fo:keep-together.within-page="1">到Determine以下白血病细胞浸润，CD45.2 <sup>+</sup> C1498细胞，B淋巴细胞，单核细胞和粒群体（包括祖细胞，前体和成熟细胞）髓造血干细胞数量是否降低，用免疫荧光染色和多参数流式细胞分析进行定量。白血病细胞代表16向造血细胞的36％（未显示数据）。在其他类型的细胞均存在于显著较低的数字中比在PBS中注射小鼠的C1498-注射的小鼠（通过5倍平均为B细胞亚群，4倍平均粒细胞和平均3倍对于单核细胞亚群）（ <strong>图5A至C）。</strong> </p><p class="jove_content" fo:keep-together.within-page="1">白血病和对照小鼠血液样品中的单核细胞的频率的一项调查表明，它们含有淋巴细胞（ <strong>图6A）</strong>的可比较的百分比，但是单核细胞的更高频率和白血病细胞。这些特征代表急性单核细胞白血病<sup>11（</sup> <strong>图6B）</strong>的。 </p><p class="jove_content" fo:keep-together.within-page="1">间急性单核细胞白血病<sup>12</sup>的另一特点，C1498注射小鼠带有肿肝脏（肝），肺和脾（脾肿大）（ <strong>图7A）。</strong>使用免疫荧光染色在这些器官进行检测和流式细胞术分析（ <strong>图7B）CD45.2</strong> <sup>+</sup> C1498细胞的不同频率。脾肿大从高数量的单核细胞浸润导致的，我们也估计脾人口的比例。在B淋巴细胞，单核细胞和粒细胞部分的数分别显著越大，由平均2倍，2.5倍和3倍，分别在白血病脾脏比对照脾脏（ <strong>图7C）。</strong> </p><p class="jove_content" fo:keep-together.wi<html>薄页="1"&gt; <img alt="图1" src="/files/ftp_upload/54270/54270fig1.jpg" /><br />首先确定<strong>图1中的协议设置为<em>在体外</em>培养的C1498细胞系和<em>在</em>急性白血病的<em>体内</em>描述表征。</strong>造血谱系和组织培养C1498细胞的分化阶段<strong>的示意图</strong> 。然后C1498细胞注射入同类系小鼠以诱导急性白血病的发展。进行骨髓，外周血，脾，肝和肺组织的隔离，以确定的频率，表型和形态变化的C1498细胞浸润后。四:静脉<strong>MGG:</strong>五月-格伦沃尔德姬姆萨<a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p><p class="jove_content" fo:keep-together.within-page="1"><html>广告/ 54270 / 54270fig2.jpg"/&gt; <br /> <strong>图2. C1498细胞表型分析<em>在体外</em>培养。</strong>代表流式细胞仪散点图和细胞表面<strong>（A）</strong>和细胞内<strong>（B）</strong>的直方图<strong>后，C1498-</strong>表示，用成熟的造血细胞分化相关分子所示。 C1498细胞从培养物中收获，洗涤并标记使用分别特异于细胞表面的CD11b，CD18和B220标记或它们的同种型对照的荧光抗体。对于细胞内染色，将细胞固定，透和使用针对的Mac-3，CD3ε抗体和TCR（T细胞受体）Vβ链或它们的同种型对照的共同表位的标记。使用带活细胞设门进行分析。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p><html> ntent"FO:保together.within页="1"&gt; <img alt="图3" src="/files/ftp_upload/54270/54270fig3.jpg" /><br /> <strong>图3.功能和培养C1498细胞的形态学特征</strong> 。从培养物收获并离心在玻片上进行显微镜C1498细胞<strong>。（A）中</strong> ，使用免疫荧光进行用于髓过氧化物酶表达染色<strong>。（B）中</strong>的细胞化学反应用于分析α萘丁酸酯酶（NBE）和萘酚AS-D氯乙酸酯酶（CAE）的C1498细胞活动。细胞被认为是阳性时褐色和红色紫色，大细胞质颗粒，分别观察每个标签<strong>。（c）</strong>可以-Grünwald的吉姆萨（MGG）C1498细胞染色。对于每个染色实验中，显微镜物镜放大倍率被表明。每个图像是代表三个独立的实验。large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 </p><p class="jove_content" fo:keep-together.within-page="1"><img alt="图4" src="/files/ftp_upload/54270/54270fig4.jpg" /><br /> <strong>图4.骨髓形貌在PBS-和C1498注射小鼠骨髓</strong>细胞从PBS-和C1498细胞注射的小鼠中分离，离心到幻灯片显微镜<strong>（A）</strong>可以-Grünwald的吉姆萨（MGG）染色<strong>。（B ）α</strong>萘丁酸酯酶（NBE）和AS-D氯乙酸酯酶（CAE）的功能是使用细胞化学评价<strong>（C）的</strong>萘酚。在面板A中，带（未成熟）或分段（成熟）嗜中性粒细胞是在比PBS注射的小鼠的C1498-注射的小鼠的骨髓中不可见。面板B和C表明，有相对于在对照骨髓中观察到的数字单核和粒细胞在白血病骨髓的积累。所有用放大100倍的目标进行微观分析。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p><p class="jove_content" fo:keep-together.within-page="1"><img alt="图5" src="/files/ftp_upload/54270/54270fig5.jpg" /><br /> <strong>图5.髓质种群PBS-和定量分析C1498注射小鼠骨髓</strong>细胞从PBS-和C1498细胞注射的小鼠中分离，并进行细胞计数后估计。免疫染色和流式细胞仪分析活细胞门控流之后，确定了不同的细胞群的频率<strong>。（A）中</strong>的B细胞亚群包括CD19 <sup>+</sup> B220 <sup>+</sup>细胞从原B细胞阶段到成熟B淋巴细胞<strong>（B）的</strong>粒细胞中的CD3 <sup>-</sup>和CD11b <sup>+</sup> Ly6G <sup>+</sup>谱系，包括前体的。第二未成熟和成熟粒细胞<strong>（C）</strong>中的单核细胞亚群被定义为CD3 <sup>-</sup> CD115 <sup>+</sup>和包含在祖细胞到成熟单核细胞的阶段。 N = 7只小鼠/组，并且数据表示为直方图示出了平均值±SEM表示。 ***，P &lt;0.0001和**，P &lt;0.01，非配对<em>t</em>检验比较PBS-C1498和注射小鼠。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p><p class="jove_content" fo:keep-together.within-page="1"><img alt="图6" src="/files/ftp_upload/54270/54270fig6.jpg" /><br /> <strong>在PBS-和单核细胞亚群图6.血液分析C1498注射小鼠</strong> 。代表流式细胞仪已分别定义为CD3 <sup>+</sup>和B220 <sup>+</sup>细胞在PBS-C1498和<strong>（一）T，B</strong>淋巴细胞百分比，散点图细胞注入以及CD115 <sup>+</sup>的Ly6C <sup>高</sup>细胞<sup>-</sup>小鼠<strong>（B）</strong>以C1498白血病和控制单核细胞的细胞的频率（PBS）的小鼠通过分析CD115 <sup>+</sup>的Ly6C确定。通过门控活细胞进行分析。为了比较白血病和对照组，CD45.2 <sup>+</sup>细胞C1498排除。 <a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p><p class="jove_content" fo:keep-together.within-page="1"><img alt="图7" src="/files/ftp_upload/54270/54270fig7.jpg" /><br /> <strong>在白血病和对照组小鼠脾种群图7.估计。 （A）的</strong>肝，肺和脾的代表性照片在白血病小鼠肿胀相比对照小鼠。收集脾，称重，脾细胞计数以下组织破坏<strong>。（B）</strong>柱状图表示差异白血病细胞频率erent器官<strong>（℃）</strong>后，对CD45.2 <sup>+</sup>细胞进行免疫染色，并使用流式细胞仪进行分析的结果。脾B，免疫染色后粒和单核细胞数的估计和流式细胞仪分析选通进行，以确定现场CD19 <sup>+</sup> B220 <sup>+</sup> ，CD3 <sup>-</sup>细胞CD11b <sup>+</sup> Ly6G <sup>+，CD3</sup> <sup>-</sup>细胞CD11b <sup>+</sup>的Ly6C <sup>-</sup>和CD3 <sup>-</sup>细胞CD11b <sup>+</sup>的Ly6C <sup>高</sup>细胞。对于肺，脾脏和肝脏中所示的比例条表示1厘米。 。每组5 - 8只小鼠/组，和该数据被表示在直方图为平均值±SEM <em>* P &lt;0.05;</em> <em>**，p</em>值= 0.0033，非配对<em>t-</em>检验比较PBS-和C1498注射小鼠<a href="http://ecsource.jove.com/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p><table fo:keep-together.within-page="1"><tbodY&gt; <tr><td> <strong>电池类型</strong> </td><td> <strong>膜或细胞内分子</strong> </td></tr><tr><td></td><td></td></tr><tr><td> <strong>前体和成熟细胞</strong> </td><td></td></tr><tr><td> NK细胞</td><td> NK1.1 +，泛NK + </td></tr><tr><td> NKT细胞</td><td> NK1.1 +，泛NK +，TCR Vbeta +（8.2），CD3 + </td></tr><tr><td> T淋巴细胞</td><td> TCR Vbeta +，CD3 +，CD4 +，CD8 + </td></tr><tr><td> B细胞的前体，B淋巴细胞</td><td> B220 +，CD19 +，CD21 / 35 + </td></tr><tr><td>粒前体和粒细胞</td><td> Ly6G + MAC-1 +，+的CD11b </td></tr><tr><td>单核细胞前体和单核/巨噬细胞</td><td>的CD11b + MAC-1 +，苹果-3 +，CD21 / 35 +，CD115 +，Ly6Chi </td></tr><tr><td></td><td></td></tr><tr><td> <strong>先觉</strong> </td><td></td></tr><tr><td>多能祖细胞</td><td> CD117 +的Sca-1 + CD34 +（Lin-的CD150-） </td></tr><tr><td>淋巴引发的多潜能祖细胞</td><td> CD117hi的Sca-1hi CD127 +（Lin-的） </td></tr><tr><td>常见的淋巴祖</td><td> CD117lo的Sca-CD127 1LO +（Lin-的） </td></tr><tr><td>共同髓系祖细胞</td><td> CD16 / CD117 32lo + CD34int（Lin-的的Sca-1） </td></tr><tr><td>粒细胞 - 巨噬细胞祖细胞</td><td> CD16 / CD117 32hi + CD34hi（Lin-的的Sca-1） </td></tr><tr><td>巨核细胞祖红</td><td> CD16 / CD117 32lo + CD34lo（Lin-的的Sca-1） </td></tr><tr><td></td><td></td></tr><tr><td> <strong>造血干细胞</strong> </td><td> CD117 +的Sca-1 + CD150 +（Lin-的CD34-） </td></tr></tbody></table><p class="jove_content" fo:keep-together.within-page="1"> <strong>表1.造血细胞谱系和分化的标志物。</strong> <br /> CD:分化簇;林:成熟细胞的标记;罗:低表达;喜:高表达; INT:中间表达; NK:自然杀伤细胞; TCR:T细胞受体。 </p>

Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

<p class="jove_content">该原稿提供了可用于表征<em>在体外</em>和诱导小鼠的注射后急性白血病C1498细胞培养技术的过程。表型和功能分析使用流式细胞仪，免疫荧光显微镜，细胞化学和五月 - 格伦沃尔德姬姆萨染色进行。 </p>

详细的定性C1498小鼠细胞系及其关联白血病小鼠模型协议

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

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Research

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Cancer Research

20.0K 次观看.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC). 该原稿提供了可用于表征在体外和诱导小鼠的注射后急性白血病C1498细胞培养技术的过程。表型和功能分析使用流式细胞仪，免疫荧光显微镜，细胞化学和五月 - 格伦沃尔德姬姆萨染色进行。 

视频: A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Evolutionary Relationships

<p>Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4<sup>th</sup> century BC. Aristotle&#8217;s system was improved upon during the Renaissance and then, subsequently, by Carolus Linnaeus in the mid 1700&#8217;s. These more formal classification and organization systems grouped species by their physical similarity to one another. For example, all vertebrates have a backbone, but invertebrates do not. Traits like the backbone are called synapomorphies, which are traits that are shared by a group of organisms, presumably because they were derived from a common ancestor. As we will explore, this method has been shown to have limitations and has more recently been amended to include genetic analysis. Still, scientists construct trees called dendrograms to create a visual representation of how species are related to one another and share common ancestors. These dendrograms can aid in our understanding of the evolutionary processes that drive these relationships. Genetic comparisons have added an important tool guiding the analysis of evolutionary relationships.</p>
<h4>Types of Dendrogram</h4>
<p>A type of dendrogram, called a cladogram, depicts the hypothetical genealogical relationships between species with the tips (or leaves) of the tree representing a species and the branches showing how species are related to each other. A slightly more complicated type of tree, called a phylogram, differs from a cladogram in that the branches leading to the species are of different lengths. The length of a branch in this type of tree represents the degree of change between species: the longer the branch, the more time since the species have diverged from a common ancestor. In both types of tree, the common ancestor of a group of species is indicated by a node, which is the point where a series of branches meet. Species that are more closely related to each other (most recently shared a common ancestor) will be located closest to the node. The two species that share a node are called a sister group<sup>1</sup>.</p>
<h4>Understanding Evolutionary Relationships using Genetic Data</h4>
<p>Historically, cladograms were constructed by comparing the morphology (physical structure) of organisms. This method is still practiced but the techniques have been modernized to include comparison of DNA (deoxyribonucleic acid) sequences between species. Using DNA for building trees has several advantages over relying solely on morphology, including being able to calculate an estimate of how long ago different species shared a common ancestor<sup>1</sup>. However, using DNA is not always feasible, especially when trees include extinct organisms. DNA is best found in soft tissues, which are not preserved during the fossilization process, and therefore it is uncommon for a DNA sample of an extinct species to be available.</p>
<p>DNA is passed on from parents to their offspring in hereditary units called genes. The nucleotide (A, G, C, and T) sequence of genes found in different species are frequently quite similar, likely due to their having come from a common ancestor. This fact allows researchers to align sequences from different species with one another to build the trees described above. Species with more similarity between their nucleotide sequences will be placed next to each other in a tree, and species with less sequence similarity will be placed further apart from each other.</p>
<p>Bioinformatics are the tools used by biologists to analyze large datasets using a combination of computer science, mathematical modeling, and statistics. One such tool is called BLAST (Basic Local Alignment Search Tool), which can be used to quickly search the entire genome of any species that is available in the NCBI (National Center for Biotechnology Information) database<sup>2</sup>. The NCBI database combines several different databases that hold different types of DNA sequence information. The process of a BLAST search includes complex computer algorithms, but basically, BLAST aligns sequences of each nucleotide base from a submitted DNA sequence (known as the <em>query</em> sequence) with sequences in the data base that most closely match it. The DNA sequences that are found will be listed in order of similarity to the sequence in question, and will therefore be from species closely related to the species containing the query gene. This comparison may or may not depict the actual evolutionary relationship between species because genes evolve at different rates. Additionally, genomes sometimes contain more than one instance of a similar sequence.</p>
<p>Comparison of DNA sequences of genes is valuable beyond consideration of evolutionary relationships. Frequently, genes are identified in model organisms, such as the fruit fly, <em>Drosophila melanogaster</em>, or the mouse<sup>3</sup>. Integral to studying a gene, the function of its product is commonly identified and analyzed. If a researcher is interested in studying that function in a different organism (humans for example), BLAST or other bioinformatic tools can be used to find candidate genes based on their similarities to the genes of known function from model organisms.</p>
<p>Human genes can also be used as the starting point to find homologs in model organisms. In fact, human disease research depends heavily on this. Once a human gene of interest is identified, mice can be genetically manipulated to have the homologous gene disrupted, or &#8220;knocked out,&#8221; creating a model of the human disease that can be studied in order to understand and treat the disease. There are many of these mouse strains currently available. For example, there is a mouse model for human Cystic Fibrosis (CF) called the <em>Cftr</em> knockout mouse and another modeling atherosclerosis, called the <em>Apoe</em> knockout<sup>3</sup>.<br />
 </p>
<h4>References:</h4>
<ol class="references">
	<li><strong>Annenberg Learner.</strong> Online Textbook: Unit 3 Evolution and Phylogenetics. <em>Rediscovering Biology. </em>[Online] 2017. [Cited: August 23, 2018.] https://www.learner.org/courses/biology/textbook/compev/compev_3.html.</li>
	<li><strong>National Center for Biotechnology Information, U.S. National Library of Medicine.</strong> BLAST: Basic Local Alignment Search Tool. [Online] https://blast.ncbi.nlm.nih.gov/Blast.cgi.</li>
	<li><strong>National Human Genome Research Institute.</strong> Background on Mouse as a Model Organism. <em>National Human Genome Research Institute. </em>[Online] December 2002. [Cited: August 23, 2018.] <a href="https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/" target="__blank">https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/</a>.</li>
</ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Concept

进化关系

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system ...

教育

JoVE Lab Manual

生物学

进化

Concentration Dependence

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Concentration Dependence<button class="expand">Expand</button>
	<p>In this experiment, you will combine varying concentrations of aqueous HCl and sodium thiosulfate to make solid sulfur, which rapidly gathers into opaque yellow particles at a certain sulfur concentration. Since the reaction solution starts clear and colorless, you can easily tell when it reaches that concentration.</p>
	<p>You&#39;ll use the same test tube and total volume each time, so it will take the same amount of sulfur to make each solution completely opaque. Thus, you&#39;ll measure the reaction progress by timing how long that takes. You&#39;ll then use that data to estimate the reaction orders of the individual reactants and of the overall reaction.</p>
	<p>Before starting the lab, make a table listing the reactant concentrations, the time to solution opacity, and the solution temperature for the trials.</p>
	<h4><strong>Table 1: Reaction progress</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td><strong>Trial</strong></td>
				<td><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] (M)</strong></td>
				<td><strong>[HCl] (M)</strong></td>
				<td><strong>Time (s)</strong></td>
				<td><strong>Temp (&#176;C)</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">2</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">3</td>
				<td style="text-align: center;">0.2 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">4</td>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">5</td>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">6</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">6.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">7</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">4.5 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">8</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">1.5 M</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_1._Reaction_progress.xlsx" target="_blank">Click Here to download Table 1</a></p>
	<p>We want to make sure that the reactions are all taking place at room temperature. You&#39;ll perform two benchmark trials, three trials with different sodium thiosulfate concentrations, and three trials with different HCl concentrations. You will vary the concentrations by diluting stock solutions of sodium thiosulfate and HCl, as shown in the following tables.</p>
	<h4><strong>Table 2: Sodium thiosulfate dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 0.2 M sodium thiosulfate</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">5 mL</td>
				<td style="text-align: center;">15 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.10 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">10 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">15 mL</td>
				<td style="text-align: center;">5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.20 M</td>
				<td style="text-align: center;">20 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_2._Sodium_thiosulfate_dilutions.xlsx" target="_blank">Click Here to download Table 2</a></p>
	<h4><strong>Table 3: HCl Dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 6.0 M HCl</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1.5 M</td>
				<td style="text-align: center;">2.5 mL</td>
				<td style="text-align: center;">7.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">3.0 M</td>
				<td style="text-align: center;">5.0 mL</td>
				<td style="text-align: center;">5.0 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">4.5 M</td>
				<td style="text-align: center;">7.5 mL</td>
				<td style="text-align: center;">2.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">6.0 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_3._HCl_Dilutions.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<p>Remember to use caution when handling HCl, which is toxic and highly corrosive. Sulfur dioxide, which is a gaseous product of this reaction, is also toxic. You will leave the reaction waste in the fume hood overnight to let the sulfur dioxide escape harmlessly.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-6">
		<div class="lab-step">First, put on a lab coat, splash-proof safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Fill a plastic wash bottle with deionized water and get a test tube marked with an X.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Clamp the test tube over the center of a small stir plate. The label must be on the opposite side from you. Make sure that you can clearly see the X. Place a small magnetic stir bar in the test tube.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use a thermometer clamp to secure a glass thermometer in the lower third of the tube, keeping it clear of the X and the stir bar.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Cut a sheet of about 20 squares of plastic paraffin film from a roll and bring the sheet back to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Lay paper towels in the fume hood as a clean surface for labware to be reused.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Label a 600-mL beaker as &#8216;waste&#8217; for the reaction, a 100-mL beaker as &#8216;0.2 M sodium thiosulfate&#8217;, and a 50-mL beaker as &#8216;6 M HCl&#8217;.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Fill the 100-mL beaker with 0.2 M sodium thiosulfate from the stock bottle in the dispensing hood.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">After that, bring the 50-mL beaker and a watch glass to the acid-dispensing hood to obtain 50 mL of 6 M HCl. Cover the filled beaker with the watch glass and bring your portion of HCl back to your hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Prepare 20 mL of 0.1 M sodium thiosulfate for the first benchmark trial. Use a 10-mL volumetric pipette to measure 10 mL of 0.2 M sodium thiosulfate and dispense it into a 20-mL volumetric flask. Fill the 20-mL volumetric flask with deionized water to the mark.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Then, cut a square of plastic paraffin film, seal the flask, and invert it several times to thoroughly mix the solution.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Remove the film and pour the solution into the test tube.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Prepare 10 mL of 3 M HCl. Pipette 5 mL of 6 M HCl into a 10-mL graduated cylinder. Add deionized water to fill the graduated cylinder to the 10-mL line.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Seal the top of the graduated cylinder with plastic paraffin film and thoroughly mix the HCl solution by inverting it several times.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, start the stir motor and get a reset stopwatch. Confirm that you can see the X through the solution in the test tube.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Simultaneously start the stopwatch and pour the 3 M HCl into the test tube.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Watch the X through the solution, which will start becoming cloudy when enough sulfur has been produced to saturate it. Stop the stopwatch exactly when the solution becomes so opaque that the X disappears.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Record the time in your lab notebook for benchmark trial 1 and then reset the stopwatch. Also, record the temperature of the solution.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Now, turn off the stir motor, remove the thermometer, and rinse it with deionized water into the waste beaker.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Then, empty the test tube into the waste beaker, retrieve the stir bar with long forceps, and rinse the test tube and stir bar with deionized water.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Thoroughly clean the test tube with a test tube brush. Briefly rinse the volumetric flask and graduated cylinder with deionized water as well. Dry everything with paper towels.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Repeat the trial in the exact same way for the second benchmark time. If the time is not within 3 - 5 s of the first trial, repeat the benchmark trial until you have two consistent trials.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Follow the same overall procedure for each subsequent trial, changing the dilutions as indicated in your table and cleaning your glassware between each trial. Use a 5-mL volumetric pipette as needed when measuring the sodium thiosulfate stock solution in trials four and five.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once you have performed all eight trials, rinse your glassware and tools one last time and neutralize the reaction waste with baking soda. Use caution because sulfur dioxide is one of the reaction products.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Transfer the neutralized reaction waste to the designated waste container. Then, pour any remaining sodium thiosulfate solution into its labeled waste container and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Neutralize any remaining HCl with baking soda before flushing it down the drain with tap water.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Finally, clean and put away your labware according to your standard practices and throw out any trash remaining in your hood. Your instructor will dispose of the reaction waste later.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Now, estimate the reaction order of sodium thiosulfate and HCl. Calculate the starting concentration of sodium thiosulfate in the reaction mixture for trials 1 through 5. Looking at the concentration of sodium thiosulfate and the reaction times, we see that as the concentration doubles, the time to opacity is halved.
		<h4><strong>Table 4: Estimating reaction order sodium thiosulfate</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] added</strong></td>
					<td style="text-align: center;"><strong>Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> volume (mL)</strong></td>
					<td style="text-align: center;"><strong>Total volume (mL)</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
					<td style="text-align: center;"><strong>Inverse time (s<sup>-1</sup>)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_4._Estimating_reaction_order_sodium_thiosulfate.xlsx" target="_blank">Click Here to download Table 4</a></div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Remember that each trial used the same volume, so it took the same amount of sulfur to make each solution opaque. Thus, the reaction is twice as fast when the sodium thiosulfate concentration is doubled.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">When the concentration increases by a factor of 4, the reaction rate also increases by a factor of 4. This one-to-one relationship suggests that the reaction was first order for sodium thiosulfate. If so, we would expect a plot of rate versus sodium thiosulfate concentration to be linear.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">We didn&#39;t measure how much sulfur it took to turn the solution opaque, but we assume that it was the same for each trial. This lets us use the inverse of time to opacity as a stand-in for the rate. The slope won&#39;t be equal to k, but that&#39;s OK for this lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Plot inverse reaction time as a function of sodium thiosulfate concentration. This confirms the linear relationship between sulfur production and thiosulfate concentration. Thus, the reaction seems to be first order with respect to thiosulfate.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the same method to estimate the reaction order of HCl. First, calculate the starting concentration of HCl in the benchmark trials and in trials 6 through 8.
		<h4><strong>Table 5: Estimating reaction order HCl</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[HCl] added</strong></td>
					<td style="text-align: center;"><strong>HCl volume (mL) </strong></td>
					<td style="text-align: center;"><strong>Total volume (mL) </strong></td>
					<td style="text-align: center;"><strong>[HCl] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_5._Estimating_reaction_order_HCl.xlsx" target="_blank">Click Here to download Table 5</a></div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Then, look at the concentration of HCl and the reaction times for these trials. The reaction times are nearly identical, so the HCl concentration has no effect on the reaction rate. This means that HCl seems to be zeroth order for this reaction.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Lastly, add up the reaction orders of the reactants to get the overall reaction order.</div>
		</li>
	</ul>
	</li>
</ol>

Concentration Dependence and Reaction Order - Procedure

浓度依赖性

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Concentration DependenceExpand
	In this experiment, you will combine varying concentrations of ...

化学

Lab Manual Sub Articles

Hardy-Weinberg and Genetic Drift

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Class Simulation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Begin by opening a new spreadsheet file. Following the Hardy-Weinberg equation where p is the frequency of a dominant allele A in a population, and q is defined as the frequency of a recessive allele B, input frequency p of allele A into cell B2, and frequency q of allele B into cell B3.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Assign the value 0.5 to cell C2.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Following the 1 &#8211; p = q equation, enter the formula &#8220;= 1 &#8211; C2&#8221; into cell C3 to calculate the frequency q of allele B. NOTE: Cells C2 and C3 will represent the gene pool used in the next few steps.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Label cells E2 and F2 &#8220;Allele 1&#8221; and &#8220;Allele 2&#8221; respectively, then enter the RAND code for random formula into cell E3. =IF (RAND()</div></li><li data-sub-note-item="1-5"><div class="lab-step">Select cell E3 and drag the bottom right corner of the cell down to E27 to duplicate the formula into 25 cells, creating an allele for the other offspring, and enter the same formula that was used in cell E3 into cell F3.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Add a third column of data starting with the description &#8220;Genotype&#8221; in cell G2, and add the CONCATENATE function to cell G3 to combine the two randomly generated alleles and to create a genotype.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Drag this formula down for 25 cells and label cells H2, I2, and J2 AA, AB, and BB respectively.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Then, input one IF function as shown here into cells H3, I3, and J3. Drag the formulas down for 25 rows per column. The formulas will return a 1 if the genotype in this row matches the genotype in the header row, and a 0 if the genotype in this row is one of the other two genotypes. H3: = IF (G3=&#8221;AA&#8221;,1,0) I3: =IF(OR(G3=&#8221;AB,G3=&#8221;BA&#8221;),1,0) J3: =IF(G3=&#8221;BB&#8221;,1,0)
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, label cell D28 as &#8220;SUM&#8221;, add the formula to cell H28, and drag the formula to cells I28 and J28 to get the total number of each genotype.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Next, label cells H30 and J30 &#8220;A&#8221; and &#8220;B&#8221; respectively and label cell D31 &#8220;Number of alleles&#8221;.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Add code to cells H31 and J31 to obtain the total number of alleles in the simulated generation, which is two times the frequency of the respective homozygous genotype, plus the frequency of the heterozygous genotype.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, label cell E32 &#8220;Next gen allele frequency&#8221; and add code to cells H32 and J32 to obtain the ratio of alleles in the next generation.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Using the gene frequencies generated in the mathematical model you just built, adjust the gene pool values in cells C2 and C3 to those from H32 and J32, and run 10 more generations, updating the gene pool with the resulting allele frequency each time.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Record the ratio of the two variables at each time point in the spreadsheet on the table, and create a line graph to see how a small population changes over time. Hypotheses: The experimental hypothesis in this study is that smaller populations will deviate more from the Hardy-Weinberg expected frequencies of the alleles than will larger populations, because smaller populations are more susceptible to genetic drift. The null hypothesis is that the allele frequencies within the populations will not differ from the Hardy-Weinberg equilibrium equation expected frequencies, meaning that the allele frequencies will be the same from generation to generation.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Set the frequency of gene A to 0.3 and gene B to 0.7, and run the model 10 more times, recording the ratio of the genes in the next generation in the table after each run.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, drag the formulas for the cells further down to alter the model to include 100 zygotes and run the model another 10 times.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">After the last run, save all of the working data to a personal storage device and exit the spreadsheet program.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Simulation for Hardy-Weinberg and Genetic Drift
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin, find a partner and then collect one bag from the instructor.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Verify that the bag contains beads of two different colors and an even number of each color. 
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Designate one color as allele capital A and the other as small a. Hypotheses: The experimental hypothesis for this simulation is that allele frequencies will not change over the 10 generations of the experiment, and Hardy-Weinberg equilibrium will be observed. The null hypothesis is that equilibrium will not be seen in the population.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Note the initial frequency in Table 1 next to generation zero. Using the Hardy-Weinberg equation, calculate the initial genotype frequencies of the population and note this in Table 1. <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_A.xlsx" target="__blank" style="display:block;margin:20px;">Click Here to download Table A</a>
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Then, discuss with your partner about the expected final allele and genotype frequencies after 10 generations of Hardy-Weinberg equilibrium. Note this next to prediction in Table 1.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Draw a pair of beads from the bag. This represents the genotype of one individual in the next generation.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Place a tally mark in the appropriate column next to Generation 1.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Now, keeping each pair of beads out of the bag every time, repeat the drawing 20 times to fill out the generation one row of Table 1.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Using the tallies, calculate the allele frequency for that generation, remembering to count both alleles in each genotype. Make a note of the new allele frequency, and then adjust the 100 beads to reflect the population after Generation 1 by adding and removing beads as necessary.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Repeat the drawing of pairs from this adjusted population for another generation of 20 picks, recording all of the observations in Table 1.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">After calculating the allele frequency for this generation, adjust the population again to set up a third generation draw.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Keep drawing, calculating allele frequencies, and adjusting the starting population until 10 generations have been recorded in total.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Violations of Hardy-Weinberg 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To test the effect of different physical and ecological scenarios on the Hardy-Weinberg equilibrium, first draw a slip of paper to determine which Hardy-Weinberg assumption you will be testing.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Make a prediction of what you think might happen in your selected scenario before beginning the experiment, and record this on the board.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">If you selected the mutation scenario, select 5 alleles at random from the set and replace them with 5 of a third color, allele capital B.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">As before, perform the simulation by pulling pairs of beads from the bag for a generation of 20 picks, recording the results.  <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_B.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table B</a>
</div></li><li data-sub-note-item="3-5"><div class="lab-step">After each generation, make adjustments to have the starting population match the new allele frequency as before.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Then, replace 5 random alleles again with the new color, and simulate the new generation for 20 picks.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">If you picked the non-random mating test, draw 1 allele at a time instead of 2 and then pair it with another of the same color pulled from the bag.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">As with the previous simulation, adjust the population to match the new allele frequencies at the end of the previous generation, and carry out the simulation for 10 generations in total.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">For the gene flow condition, begin by removing 10 alleles from the population and replacing them with 10 randomly selected alleles from another set of 100 beads.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Adjust the allele frequency and repeat the gene flow of removing 10 beads and adding 10 beads from another bag at the beginning of each generation.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">If you drew small population size, simply start each generation with 60 alleles instead of 100.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">As usual, adjust allele frequencies each generation and record 10 generations. In the selection condition, draw from the original bag of beads but do not count any homozygous recessive pairs that are drawn when calculating the allele frequency at the end of each generation.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Simulation of Genetic Drift
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">Make your predictions before running the simulations and record them on the board.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">If you have the small population size test, run the standard Hardy-Weinberg simulation as before, but instead start each generation with 30 alleles and not 100.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">If you drew the founder effect with two alleles test, start generation 1 by drawing only 5 pairs of beads from the bag.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Then, creating the gene pool for generation 2, include only 50 beads at the allele frequency determined in generation 1. When you create the gene pool for generation 3 and all subsequent generations, include 100 beads.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">For a founder effect with 10 alleles, you will need to collect a bag containing random amounts of 10 different colored beads.
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Designate an allele name for each of the new alleles, and then calculate their initial allele frequency.
</div></li><li data-sub-note-item="4-7"><div class="lab-step">Now, run the experiment in the same way as the founder effect with two alleles, by drawing just 5 pairs randomly for generation 1 and then creating the second generation with 50 alleles, and third and subsequent generations with 100. <a href="https://www.jove.com/CDNSource/lm/labs/4/Lab_4_Table_C.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table C</a>
</div></li><li data-sub-note-item="4-8"><div class="lab-step">If you have the natural disaster with 2 alleles, you will need to collect a paper plate from your instructor.
</div></li><li data-sub-note-item="4-9"><div class="lab-step">Draw a line down the middle of the plate. Before generation 1, pour out all of the beads from a standard 100 bead setup onto the plate, and mix them randomly, spreading them evenly.
</div></li><li data-sub-note-item="4-10"><div class="lab-step">Choose one side of the plate and take all of the beads from that side. You should draw from these to create generation 1.
</div></li><li data-sub-note-item="4-11"><div class="lab-step">For generation 2 and beyond, adjust the population and use a total of 100 beads.
</div></li><li data-sub-note-item="4-12"><div class="lab-step">Finally, if you selected the natural disaster with 10 alleles test, you will need to collect a bag containing 10 different colored beads.
</div></li><li data-sub-note-item="4-13"><div class="lab-step">Assign a name to each new allele, and then place them on the plate and select half. Draw from these 50 beads for generation 1, and then calculate the allele frequencies to make a population of 100 in each subsequent round.
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">Using the entered functions, calculate the number of each allele by multiplying the number of the homozygous genotypes by 2 and adding the number of the heterozygous genotypes.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">The frequency can then be calculated by dividing the number of the allele in question by the total number of alleles. Enter the results into the table.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Next, use the Hardy-Weinberg equation to calculate the expected genotype frequency of the next generation, and compare the results of the simulation with the expected frequency, using the table as a guide.
</div></li><li data-sub-note-item="5-4"><div class="lab-step">Plot the changes of the population gene frequencies over time that you recorded in the table. Note any changes in allele frequencies over time and if any alleles decrease in frequency to the point that they disappear from the population. 
</div></li><li data-sub-note-item="5-5"><div class="lab-step">Plot the changes in the populations of the 25 and 100 zygotes with initial gene frequencies of 0.5 for each allele. Compare the results of the smaller population and the larger populations.  NOTE: The observed allele frequencies are expected to differ slightly from the Hardy-Weinberg expected values as a result of chance from the RAND function.
</div></li><li data-sub-note-item="5-6"><div class="lab-step">Using separate lines for each allele, graph the allele frequencies found for the original Hardy-Weinberg experiment over 10 generations.
</div></li><li data-sub-note-item="5-7"><div class="lab-step">Compare the results across the class, and see if your results agree.
</div></li><li data-sub-note-item="5-8"><div class="lab-step">Next, again using one line per allele, graph the results of your second experiment testing violations of the Hardy-Weinberg equilibrium, and share your finding with the class. Compare your actual results with your prediction. 
</div></li><li data-sub-note-item="5-9"><div class="lab-step">Finally, graph the results of your genetic drift simulation scenario in the same manner.
</div></li><li data-sub-note-item="5-10"><div class="lab-step">Again, present your results to the group and hypothesize why your results did or did not match your prediction. Compare your results to those of your classmates&#8217;. 
</div></li></ul></li></ol>

Hardy-Weinberg Equilibrium Principle and Genetic Drift - Procedure

Hardy-Weinberg 和遗传漂变

Class Simulation
ExpandBegin by opening a new spreadsheet file. Following the Hardy-Weinberg equation where p is the frequency of a dominant allele A in a ...

Bacterial Transformation

<ol class="note-items"><li data-note-item="1" data-parent-collapse=""><strong>Bacterial Transformation</strong>
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">IMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face away from suspension cultures, and to avoid inhaling reagents. Do not touch your face while performing the experiment. Always wash your hands before and after every experiment.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">When you are ready to safely begin, check to make sure you have a tube or tubes of competent <em>E. coli</em> cells in your ice bucket.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, transfer 50 &#181;L from one of these tubes to each of two new micro-centrifuge tubes, one labeled '&#8211;', and one labeled '+'.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, pipet 2 &#181;L of p-GREEN plasmid into the plus tube and flick the tube gently to incorporate the plasmid throughout the bacterial solution. HYPOTHESES: The experimental hypothesis is that on plates containing no antibiotic, bacteria with or without the plasmid can survive, and so both will grow lawns of bacteria. Conversely, on the plates containing the ampicillin antibiotic, only the bacteria successfully transformed with the resistance plasmid will grow, whereas the minus plasmid plates will contain no bacteria. The null hypothesis of this experiment is that there will be no difference in bacterial growth between the plates.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Incubate both tubes on ice.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After 30 minutes submerge both tubes 3/4 of the way into the water of a 42 &#176;C water bath for 30 seconds.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">At the end of the water bath incubation, place the tubes back on ice for 5minutes, then add 950 &#956;L of room temperature SOC media to each tube.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Incubate the tubes in a shaker at 37 &#176;C for 60 minutes at 250 rpm.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">While the tubes are incubating, label the bottoms of one LB and one LB/ampicillin, or AMP plate, &#8220;+ plasmid&#8221;, and the bottoms of one LB plate and one LB/AMP plate &#8220;- plasmid&#8221;.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">At the end of the incubation, pipet 50 &#956;L from the &#8220;+ plasmid&#8221; cell tube onto both the LB/AMP plus plate and the LB plus plate.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Then, with a fresh tip, add 50 &#956;L of the bacteria that were not exposed to the plasmid to each of the minus plates.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, spread each plate using a fresh pipette tip.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Cover the plates with their lids, and then seal them with laboratory film.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then store the plates upside down in a 37 &#176;C incubator for 24 hours.
</div></li></ul></li><li data-note-item="2" data-parent-collapse=""><strong>Results</strong>
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">The day after the transformation, observe which plates have bacteria, and how the bacteria are distributed over each plate.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Record the number of colonies and the color of the colonies for different conditions. <a href="https://www.jove.com/CDNSource/lm/labs/11/Table_1_Bacterial_Transformation.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-3"><div class="lab-step">To determine the initial mass, IM, of the plasmid, first multiply the plasmid concentration from the stock tube of p-GREEN by the volume transferred into the experimental tube.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">To calculate how much DNA is spread onto each plate, divide the volume of bacterial suspension spread onto each plate by the volume of the suspension in the tube, and multiply this fraction by the initial mass of plasmid used.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">To determine the transformation efficiency, count all of the visibly distinct colonies on the plus plasmid LB/AMP plate, and divide this number by the mass of plasmid spread. NOTE: The unit for transformation efficiencies is cfu/&#956;g of plasmid DNA.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Record all of the results for each plate in the table and analyze them.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Observe any differences between the colonies and make hypotheses to explain this observation. Consider the factors that may affect the transformation efficiency of competent bacteria.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">NOTE: You will have also noticed that each plate had a different outcome. The minus plasmid and plus plasmid LB plates should have grown a lot of bacteria across their plates. The minus plasmid LB/AMP plate should have no bacterial growth. And the plus plasmid LB/AMP plate should have grown bacterial colonies that fluoresced green.
</div></li></ul></li></ol>

Bacterial Transformation using Plasmids - Procedure

细菌转化

Bacterial Transformation
ExpandIMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face ...

减数分裂

Animal Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Making Choice Chambers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the bottom of both bottles. One line should be 1 cm from the base of the bottle and the other should be 1.5 cm from the base of the bottle.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a razor blade to make a small cut in each bottle at the mark. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, insert the scissors and carefully cut along the line until the bottoms of the bottles are removed completely.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Fit one bottle bottom into the other.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Tape the bottles together to make a secure chamber.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Use the razor blade and scissors to poke a hole into the center of the chamber. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Take a piece of tape and fold the edge onto itself to make a tab and place the tape over the hole.  Note: The tab helps with easy removal and reattachment of the tape to the chamber.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Use the Sharpie to label one side of the chamber A and the other side B.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Upon receiving the colony of <em>Drosophila</em>, perform a count to make sure there are 40 living individuals in the vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Geotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To perform a geotaxis assay, first attach a clean, empty bottle cap to each end of the choice chamber and turn the fly vial upside down onto its lid.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Open the tape flap in the center of the choice chamber.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Insert the funnel into the hole and tap all of the flies into the chamber. HYPOTHESES: The alternate hypothesis is that more flies will be moving against gravity, or up, regardless of the orientation of the choice chamber. Alternatively, if the flies are positively geotactic, then more flies will move down, or with gravity. The null hypothesis is that the flies will have no preference with regard to gravity and will move randomly within the chamber. 
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Once the <em>Drosophila</em> are in the chamber, close the tape flap, rotate the chamber so that it stands vertically with side A on the bottom, and start the timer.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Immediately, count the number of <em>Drosophila</em> that are moving up and the number that are moving down. Record these data for time point 0 in the table. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">After 30 seconds, rotate the chamber so that side B is on the bottom and count the number of <em>Drosophila</em> that are moving up or down. Record these data for time point 30 in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After 60 seconds, rotate the chamber so that side A is on the bottom again and record the number of moving flies for time point 60.  <a href="https://www.jove.com/CDNSource/lm/labs/15/Table_1_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Hold the chamber over the fly vial and carefully open the cap to return all of the <em>Drosophila</em> back to their empty vial. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Phototaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To perform a phototaxis analysis, first cover side A of the choice chamber with tin foil, taking care that the foil reaches to the exact center between the two sides. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Ensure the caps are secured on both ends of the chamber.  HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found in the light side of the choice chamber, if the flues are attracted to light, or on the dark side if they are repelled by light. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus. Therefore, equal numbers of flies will be found on either side of the choice chamber.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Open the tape flap in the center of the choice chamber, insert the funnel into the hole, and tap all 40 flies into the chamber as previously described.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Set the timer to sound an alarm at 10 minutes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">When the alarm sounds, stop the timer, and count the number of flies in the open side B of the chamber.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Subtract that number from the total number of flies added to the choice chamber to calculate the number of flies in the covered side A.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record these data in the table for time point 10.  <a href="https://www.jove.com/CDNSource/lm/labs/15/Table_2_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Carefully, return all of the flies back to their empty vial.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Chemotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found on the side of the chamber with the apple cider vinegar if the flies are attracted to the odor. Or, if the flies are repelled by the vinegar, then more of them will be found on the side of the chamber presenting the odorless water. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus and will move randomly in the chamber.  Use an individual disposable plastic pipette to dispense two drops of the apple cider vinegar onto one cotton ball for side A of the chamber. 
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Use an individual disposable plastic pipette to dispense two drops of the distilled water onto a cotton ball for side B of the chamber. 
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Fold two pieces of tape over themselves to create individual pieces of double-sided tape and use the tape to attach each cotton ball to the inside of the single bottle cap.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Screw the bottle caps with the cotton balls onto their corresponding sides of the chamber.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Transfer all of the flies into the chamber and set the timer to sound an alarm after 10 minutes. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">When the alarm sounds, stop the timer and record the number of flies in each side of the chamber. <a href="https://www.jove.com/CDNSource/lm/labs/15/Table_3_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="4-7"><div class="lab-step">At the end of the final experiment, funnel the flies into a morgue tube containing 70% ethanol. 
</div></li><li data-sub-note-item="4-8"><div class="lab-step">Dismantle the choice chambers for recycling.  NOTE: Since all of the chemicals used in these experiments are lab safe, the liquids can be disposed of down the sink, the chambers rinsed, and the solids can be thrown away in the trash. 
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">Gather all of the data collected for the geotaxis, phototaxis, and chemotaxis experiments.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Plot these values as bar graphs for different conditions and different time points.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Use these data to draw inferences about the behavior of <em>drosophila melanogaster</em>. 
</div></li></ul></li></ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Procedure

动物行为

Making Choice Chambers
ExpandBefore beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the ...

行为

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Human Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this experiment you will prepare different types of cells from a plant and animal, onion and human respectively, and then visualize them under a microscope. HYPOTHESES: The experimental hypothesis is that the cells will appear different in overall shape and membrane structure, but there will be some shared similar structures, like nuclei, in both cells. The null hypothesis of the experiment is that there will be no difference in structure between the two cell types.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">First, to prepare an animal cell slide, start by pouring 30 mL of distilled water into a beaker.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, use a glass dropper to place 2 - 3 drops of the water onto the center of a clean microscope slide.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, take a clean toothpick and scrape the inside of your cheek to gather cells.  CAUTION: Do not scrape your cheeks too aggressively. The scraping should not cause discomfort or bleeding. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Put the end of the toothpick with the cheek cells onto the wetted area of the slide and mix the water and cheek cells together.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, add two drops of methylene blue staining solution to the wetted area of the slide and mix it in with a clean toothpick.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Let the mixture sit at room temperature for 3 minutes.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After 3 minutes have passed, use a glass dropper to add a drop of glycerol to the mixture.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Carefully, place a cover slip over the mix on the slide by first standing one end of the cover slip vertically and aligning it along to one side of the mixture. Then, carefully lower the other end of the cover slip down until the mixture is completely covered.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Finally, remove any excess liquid with blotting paper.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Next, prepare a slide of plant cells by placing five drops of distilled water into a clean watch glass.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, with forceps, take a thin strip of onion and place it into the water.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Apply 5 drops of safranin solution to another watch glass.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Now using a pair of forceps transfer the piece of onion from the distilled water into the safranin.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the onion to soak for 30 seconds.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, again with forceps, move the onion back into the distilled water.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, place 3 drops of glycerol onto the center of a glass microscope slide.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Using the forceps again, transfer the onion into the glycerol on the slide.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Gently place a cover slip over the onion on the slide and blot away any excess liquid with a piece of blotting paper.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you will visualize the slides you have prepared using a compound microscope. First, turn on the scope and set the objective lens to the lowest level of magnification.  NOTE: A compound microscope typically has two levels of magnification.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Place the cheek tissue slide onto the stage of the microscope and bring the cells into focus by adjusting the stage and using the coarse adjustment knob.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">When the cells are in focus, observe the individual cells and draw what you see. Repeat these observations at medium and high magnifications adjusting the focus with the fine adjustment knob. IMPORTANT: Do NOT use the coarse adjustment knob at high magnifications. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, rotate the nose piece so that the oil lens is almost in place.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Place a drop of immersion oil on top of the cover slip of the cheek cell slide.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Use the oil lens to view the cheek cells and record your observations.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Repeat these steps to view the onion plant cell, moving again from low to medium, high, and oil lenses (steps 20 &#8211; 24).
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record all of your observations and note the differences you see between animal and plant cells.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">When you are finished, place the cheek cell slide and toothpicks into a diluted bleach solution. Dispose of the cover slips into a glass waste container.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Finally, turn off the microscope and clean the lenses with lens cleaning paper.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Calculate the total magnification for each of the viewing levels. Remember to account for both the eyepiece magnification and the objective lens magnification. NOTE: Eye piece: 10X magnification, Objective lens: 4X, 10X, and 40X magnification
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, identify the structural differences between plant and animal cells using the observations made under the microscope.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Label the nuclei in both cell types and then highlight or note any differences that you see between them.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Procedure

单元结构

Visualizing Onion and Human Cells
ExpandIn this experiment you will prepare different types of cells from a plant and animal, onion and human ...

科学探究

Genetics of Organisms

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">REF and WEF Fly Genetics
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be labeled with the notation REF for red-eyed flies or WEF for white-eyed flies. The female vial should also be labeled virgin as these flies should be unmated for the purpose of this experiment.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Make sure you have around five flies in each vial. NOTE: You will perform crosses using these flies to determine if red or white eyes is dominant and if the trait is sex-linked. You should also have two fly anesthesia wands, a paint brush, magnifying glass, fly anesthetic, some empty fly vials, and a sheet of white paper.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Draw a line in the middle of the paper to make a male side and a female side.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Dip the fly anesthesia wand into the solution and insert it into the red-eyed female vial until the flies stop moving.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Then, dip the same wand into the white-eyed male vial until these flies are also anesthetized.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Tap the flies from the two vials onto the sheet - Females on one side and males on the other.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Check to make sure that all the flies from the female vial are female and likewise, confirm that the male flies are male.  NOTE: Males are slightly smaller and have a darker solid pigment on the end of their abdomen. Females are larger and have stripes rather than a solid spot.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If there are any male flies in the female vial, obtain a new vial as these females likely have sperm from males from their own strain and are unusable.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Now, move the five red-eyed females and five white-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Label this vial to indicate that this is a cross between red-eyed females and white-eyed males. HYPOTHESES: Use Punnett squares to formulate hypotheses for this experiment. Consider what you will see in a few days if the white allele is dominant and there is no effect of parental sex. What if red is dominant? Under the potential offspring genotype in each box, write the expected phenotype.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">To set up the reverse cross, again dip the fly anesthesia wand into the solution and insert it this time into the white-eyed female vial and then the red-eyed male vial until all the flies have stopped moving.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Tap the flies onto a sheet of white paper and check to make sure that all the flies from the female vial are female and likewise with the male flies.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Now, move the five white-eyed females and five red-eyed males into a new vial using the paintbrush.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Label this vial to indicate that this is a cross between white-eyed females and red-eyed males.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Finally, place the two tubes containing the new crosses into a rack for incubation at room temperature. NOTE: After about three days, larvae should be moving around in the vial.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Remove the 10 adult flies and dispose of them into the morgue tube containing 70% ethanol.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, put the tubes back in the rack and let the larvae mature until they reach the adult stage in up to two weeks.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Once all of the larvae from these crosses have matured to adulthood, anesthetize the flies in one of the tubes using the wand.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Then, on a piece of white paper, sort the flies by sex and phenotype and then record these data.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Now, repeat this process and tally the sexes and phenotypes for the second cross (steps 1 &#8211; 18).
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Finally, once you have counted all of your flies, dispose of them in the morgue vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To begin investigating your results, first compare the data you collected with the hypothesis you made using the Punnett squares.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Did your data match the simple Punnett square hypothesis where red or white is dominant and there is no effect of sex? Compare your data with other lab groups.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">If your data does not match what your Punnett squares predicted, does this indicate that the gene may be sex-linked?
</div></li><li data-sub-note-item="2-4"><div class="lab-step">If the red gene were dominant and sex-linked, when we cross red-eyed females with white-eyed males, all of the offspring would have red eyes. Assuming all of the females were homozygous for the color allele, does this match your data for the first cross?
</div></li><li data-sub-note-item="2-5"><div class="lab-step">If white were dominant and the gene were sex-linked, we would see the exact opposite. So all of the flies would have white eyes. From your data, can you conclude from your first cross whether red or white is dominant?
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When the reciprocal cross of white-eyed females and red-eyed males is performed, if the red phenotype is dominant, all of the females would be red and all of the males would be white. This would also confirm that the gene is specifically x-linked because white-eyed females could then only be produced in crosses with white-eyed males. Did your second cross produce any white-eyed females?
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Finally, if the gene were x-linked and white was dominant, crossing white-eyed females with red-eyed males would result in all of the offspring having white eyes. Does this hypothesis fit with your second cross?
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Look over your data and conclude which of the hypotheses best fits the patterns you found in your crosses. If your data does not fit with any of these, hypothesize why this might be the case.
</div></li></ul></li></ol>

Genetics of Organisms: REF and WEF Fly Genetics - Procedure

生物体的遗传学

REF and WEF Fly Genetics
ExpandNOTE: At your station, you should have four vials of flies. They will be labeled as male or female. They will also be ...

Population Growth

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Exponential Population Growth Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: In these experiments you will use computer software to simulate different types of growth models. HYPOTHESES: The experimental hypothesis might be that if we increase either R, the reproductive rate or the initial population size, NT, that this will increase the population size after 10 generations in an exponential growth model compared to unmanipulated control populations. The null hypothesis may be that populations of different R and initial population size will have the same population size as control groups after 10 generations.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To build an exponential population growth model, first open a new workbook in Excel.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Save the file to a secure location and name it &#8220;exponential growth&#8221;.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Label cell A1 &#8220;generation&#8221;, and cell B1 &#8220;population size&#8221;.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Type the number zero into cell A2. This row will represent the initial generation.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">In cell A3 type this formula: = 1 + A2
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Click on cell A3 and drag the small box in the bottom right corner to cell A12 for 10 generations.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Type the number two in cell B2. This will represent the initial population size in the model.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Label cell E1 &#8220;growth rate&#8221;, and in cell E2 type the number 0.05. This value represents the proportion of new offspring produced by each individual for each generation, or R in the exponential growth model.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">NOTE: In the classic exponential growth equation, T is the generation, and NT is the number of organisms in the current generation. NT+1 will be is the number of individuals in the next generation.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Enter the exponential growth equation in cell B3 as the formula shown here: = B2 + (1 + E2) * B2
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Now, hit enter.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Click cell B3 and drag the small square in the bottom right corner down to cell B12 to simulate 10 generations of exponential growth.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">To observe the results, select all data, along with the headers, in columns A and B.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Click &#8220;insert&#8221; and select &#8220;scatter plot with smooth lines and markers&#8221; from the toolbar. A scatter plot should appear in the sheet. TIP: If it does not, check to see if a new sheet was created in the workbook.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Label the Y axis &#8220;population size&#8221; and the X axis &#8220;generation&#8221;. Title the chart &#8220;exponential growth of a population&#8221;.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Observe how the population increases in a classic exponential growth pattern.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Next, create Table 1 labeling the &#8220;growth rate R&#8221; as the header of column A, and &#8220;population size after 10 generations&#8221; as the second column.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Record the population size at generation 10 for the growth rate of 0.05, the rate we just simulated.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, set the growth rate in cell E2 to 0.1, and repeat the steps to generate a new scatter plot.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Record the population size at generation 10.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Continue to alter the growth rate using the values in table one, noticing how the Y axis changes to accommodate the increasing values.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">After completing Table 1, set the growth rate back to 0.05 and change the initial population size in cell B2 to 100 individuals.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Record the population size at generation 10 in a new table, Table 2.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Proceed to change the initial population size using the values in Table 2. Once the table is complete, save the Excel sheet and close it.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Logistic Population Growth Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">HYPOTHESES: In a logistic growth model, the experimental hypothesis could be that populations will approach and meet the carrying capacity, and any populations with sizes greater than the carrying capacity will decrease in the subsequent generation. The null hypothesis might be that population growth will not follow a logistic S-shaped curve in the logistic models, and that generations with population sizes greater than the carrying capacity will continue to increase or stay the same in the next generation.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">To build a logistic population growth model, open a new spreadsheet in Excel.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Save the file as &#8220;logistic growth&#8221; in the same folder as before.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Then label cell A1 &#8220;generation&#8221; and B1 &#8220;population size&#8221;.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Type the number zero in cell A2. This row will represent the initial generation.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">In cell A3 type the formula: = 1 + A2
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Now, hit enter.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Now, click on cell A3 and drag the small box in the bottom right corner to cell A12 to number up to the 10th generation.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">Type the number two in cell B2 to represent the initial population size.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">Label cell E1 as &#8220;max growth rate&#8221; and cell F1 as &#8220;carrying capacity&#8221;.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Finally, set the max growth rate in cell E2 to 0.05. This value will represent the maximum growth rate the population may achieve &#8211; &#8220;R max&#8221; in the discrete logistic equation. NOTE: In the classic logistic growth equation the term K represents carrying capacity. As the population size of the current generation or NT, approaches the carrying capacity, the growth of the population begins to slow.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Now set the carrying capacity in cell F2 to 50.
</div></li><li data-sub-note-item="2-13"><div class="lab-step">Enter the logistic growth equation in cell B3 as the formula below: = B2 + ((1 + E$2) * B2) * ((F$2 - B2) /F$2)
</div></li><li data-sub-note-item="2-14"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">Drag the formula down to cell B12 to add it to all 10 generations.
</div></li><li data-sub-note-item="2-16"><div class="lab-step">To observe the results of this exercise, highlight all data and headers in columns A and B, then click &#8220;insert&#8221; and select &#8220;scatter plot with smooth lines and markers&#8221;.
</div></li><li data-sub-note-item="2-17"><div class="lab-step">Label the Y axis population size and the X axis generation.
</div></li><li data-sub-note-item="2-18"><div class="lab-step">Title the chart as logistic growth of a population.
</div></li><li data-sub-note-item="2-19"><div class="lab-step">Observe the growth of the population as it nears carrying capacity and record the generation that the population meets or exceeds carrying capacity in a new table.
</div></li><li data-sub-note-item="2-20"><div class="lab-step">Now set the max growth rate in cell E2 to 1.
</div></li><li data-sub-note-item="2-21"><div class="lab-step">Notice how the population responds when it rises over carrying capacity.
</div></li><li data-sub-note-item="2-22"><div class="lab-step">Continue to set the max growth rate to the values in Table 3 and record the generation at which the population rises two or above carrying capacity.
</div></li><li data-sub-note-item="2-23"><div class="lab-step">Now set the max growth rate back to 0.05 and set the carrying capacity to 1,000.
</div></li><li data-sub-note-item="2-24"><div class="lab-step">Notice how the graph changes as the growth approaches the carrying capacity.
</div></li><li data-sub-note-item="2-25"><div class="lab-step">Drag the formulas in A12 and B12 down to A14 and B14.
</div></li><li data-sub-note-item="2-26"><div class="lab-step">Adjust the plot to include these new data points to show when the population reaches carrying capacity.
</div></li><li data-sub-note-item="2-27"><div class="lab-step">Save the Excel sheet and close it.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Predator-Prey Model
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Hypotheses: In our third simulation of predator-prey logistics, the experimental hypothesis could be that predator populations will increase as prey populations increase and that the two populations will experience growth and decline dependent on the population of the other species. The null hypothesis may be that the two populations will show patterns of growth unrelated to the other species&#39; population size.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">To build a predator-prey model, open a new spreadsheet in Excel.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Save the file in the same folder as the other two models and name it &#8220;predator-prey model&#8221;.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Label cell A1 &#8220;time&#8221;, cell B1 &#8220;prey&#8221; and cell C1 &#8220;predator&#8221;.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">In cell A2 type zero and hit enter.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">In cell A3 type the formula below: = 1 + A2
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Now hit enter.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Drag the formula down to cell A802 as the model will require many time points to visualize changes of both populations.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Now type 100 into cell B2 to represent the initial prey population and type 25 into cell C2 as the initial predator population.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Label cell E1 &#8220;prey growth rate&#8221;, cell F1 &#8220;consumption of prey by predator&#8221;, cell G1 &#8220;conversion efficiency&#8221; and cell H1 &#8220;starvation of predator&#8221;.
</div></li><li data-sub-note-item="3-11"><div class="lab-step">Type the number 0.02 in cell E2. This value represents the growth rate of the prey species or R in the prey population model. NOTE: Here the prey population is represented as VT and the predator population as CT. The value A is the per capita attack rate of the predator species on the prey. In the predator population model, variables are introduced for predators produced per prey consumed, F or conversion efficiency as well as for the starvation rate of the predators noted as Q.
</div></li><li data-sub-note-item="3-12"><div class="lab-step">Type 0.0005 in cell F2 for the attack rate in both the prey population and the predator population model.
</div></li><li data-sub-note-item="3-13"><div class="lab-step">Then type 0.8 in cell G2 to represent the conversion efficiency, and 0.05 in cell H2 for the starvation rate in the predator population equation.
</div></li><li data-sub-note-item="3-14"><div class="lab-step">Now type the prey growth equation in cell B3 as the formula below: =B2+(E$2*B2)-(F$2*C2*B2)
</div></li><li data-sub-note-item="3-15"><div class="lab-step">Hit enter.
</div></li><li data-sub-note-item="3-16"><div class="lab-step">Then type the prey growth equation in cell C3 as the displayed formula: =C2 + (F$2*G$2*B2*C2)-(H$2*C2)
</div></li><li data-sub-note-item="3-17"><div class="lab-step">Hit enter.
</div></li><li data-sub-note-item="3-18"><div class="lab-step">Hold down the shift keys and click to select both cells B3 and C3.
</div></li><li data-sub-note-item="3-19"><div class="lab-step">Click the small box at the bottom right corner of cell B3 and drag the two formulas down to row 802 to simulate the predator and prey populations over 800 generations.
</div></li><li data-sub-note-item="3-20"><div class="lab-step">To visualize the data, select all of the data and headers in columns A, B and C.
</div></li><li data-sub-note-item="3-21"><div class="lab-step">Click &#8220;insert&#8221; and select &#8220;scatter plot with smoothed lines&#8221;.
</div></li><li data-sub-note-item="3-22"><div class="lab-step">A chart showing the population change of the predator and the prey populations should appear.
</div></li><li data-sub-note-item="3-23"><div class="lab-step">Label the Y axis of the plot population size and the X axis time.
</div></li><li data-sub-note-item="3-24"><div class="lab-step">Title the plot &#8220;predator-prey interactions&#8221;.
</div></li><li data-sub-note-item="3-25"><div class="lab-step">Observe the interactions between the predator and prey populations.
</div></li><li data-sub-note-item="3-26"><div class="lab-step">Set the initial predator population to zero and observe what happens in the plot.
</div></li><li data-sub-note-item="3-27"><div class="lab-step">Now set the initial predator population to 120 and observe what happens to the predator population when there are more predators than prey.
</div></li><li data-sub-note-item="3-28"><div class="lab-step">Finally set the initial prey population to zero and observe what happens to the population of the predators.
</div></li><li data-sub-note-item="3-29"><div class="lab-step">Experiment with other values to see what other interactions can be created.
</div></li><li data-sub-note-item="3-30"><div class="lab-step">When finished, save the workbook and close it.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">In the exponential growth model built in the first activity, observe the shape of the resulting population growth curve, and consider whether you think this type of growth could be sustained in nature.
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Note how the growth rate R affected the population size after 10 generations in the exponential growth model.
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Also consider how the initial population size affected the population size after 10 generations in the exponential growth model.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Now, consider the logistic growth model, and observe the shape of this graph, noting how it differs from the exponential growth model.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Then, note how the populations behave as they approach the carrying capacity in your logistic growth model.
</div></li><li data-sub-note-item="4-6"><div class="lab-step">Decide if you think that the maximum per capita growth rate, R max, influenced how quickly populations reached carrying capacity in the logistic growth model.
</div></li><li data-sub-note-item="4-7"><div class="lab-step">Also, consider what happens to population size when populations are over carrying capacity in the logistic growth model, or what might happen if the carrying capacity is lowered.
</div></li><li data-sub-note-item="4-8"><div class="lab-step">In the predator-prey model, note whether the prey population responded to increasing predator population. Conversely, consider how the predators then respond to decreasing prey populations.
</div></li></ul></li></ol>

Population Growth: Exponential and Logistic Growth and Predator-Prey Model - Procedure

种群增长

Exponential Population Growth Model
ExpandNOTE: In these experiments you will use computer software to simulate different types of growth models. ...

人口增长

Natural Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Natural Selection
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, cut the white, black, and gray pipe cleaners into quarters, which should produce a total of 100 pieces that are 3 inches long.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, cut the green pipe cleaners in the quantities and lengths specified in the video, taking care to minimize waste. This approximates a normal distribution of lengths and should result in an additional 100 pieces total. NOTE: One 12-inch pipe cleaner should yield multiple lengths, for example, a 5-inch piece and a 7-inch piece. Use this advantage to reduce waste.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Now, cut one sheet of black and one sheet of white construction paper into approximately 3-inch strips measured along the long edge of the paper.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Tape these strips down to cover the base of a dissecting tray to create an alternating pattern of black and white strips. NOTE: In the first simulation, you will use the white, gray, and black pipe cleaner segments against a black and white background to simulate different selection pressures due to predation on a population. You will select your prey using two different strategies, eyes-open and eyes-closed. For each of these strategies, you will perform four rounds consisting of 25 selections, alternating between two partners. After each round you will restore the population back to its original size using the proportions of phenotypes present after selection.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To begin the first scenario, count out 12 white, 12 black, and 26 gray pipe cleaner segments with your partner. Hypotheses: Here the experimental hypothesis might be that different predation strategies, such as having the eyes open or closed, will result in distinct selection pressures on the populations. Further, different phenotypes may be positively selected in the alternate scenarios. The null hypothesis might be that different predation strategies will have no effect on the populations or phenotypes.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Mix the pipe cleaner segments together as best as possible. Then, sprinkle them into the papered tray. NOTE: Ideally, the pipe cleaners will be spread out. However, this may not occur due to their self-adhering nature.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once the segments are in the tray, separate them as best as possible without moving them too far from where they landed. As few segments as possible should be touching.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Have the first partner close their eyes. When given an audible signal by the second partner, the first should open their eyes and grab the first pipe cleaner they see from the tray as quickly as possible.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Set the removed pipe cleaner to the side.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Alternating partners, repeat this process a total of 25 times (steps 8 &#8211; 9).
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Count the remaining pipe cleaners by color and record this in the data collection table for Scenario 1. <a href="https://www.jove.com/CDNSource/lm/labs/23/Lab_23_Tables_1_and_2.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Tables 1 and 2</a>
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, restore the population to 50 by adding one pipe cleaner of the same color for each of the remaining pipe cleaners. Hold these to the side until they are all gathered.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then, sprinkle the pipe cleaners into the tray as previously described (steps 6-7). Repeat this process for three more rounds consisting of 25 selections each and record the counts for each of the rounds in the data collection table for Scenario 1.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Remove all pipe cleaners from the tray after the fourth round.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For Scenario 2, again count out 12 white, 12 black, and 26 gray pipe cleaner segments and sprinkle them randomly into the tray.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Follow the same protocol as in Scenario 1. However, when given the signal, the grabbing partner should keep their eyes closed. To aid in accuracy, it may help to position the hand near to the tray before grabbing.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Repeat this process 25 times, switching partner roles, and record the remaining pipe cleaner amounts in the data collection table for Scenario 2.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, restore the population to 50 based on that count. Do this for all four rounds. NOTE: In the second simulation, you will be using the eyes-open and eyes-closed predation strategies as previously described to select pipe cleaners of various lengths.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">For Scenario 3, gather all 100 of the pipe cleaners that have been cut to varying lengths to represent different phenotypes.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">One partner should hold all the pipe cleaners upright and loosely in one hand resting on the tabletop, such that the bottom end of each pipe cleaner rests against the tabletop.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, the partner that is not holding the pipe cleaners should close their eyes. When given an audible signal, the partner should open their eyes and grab the first pipe cleaner they see from their partner&#39;s hand.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat this process 50 times (step 21). However, the partners should not switch roles during the round this time. The partner holding the pipe cleaners should always be the one giving the signal and never grabbing from the bunch.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">After the first round consisting of 50 selections, count the remaining pipe cleaners by length. Then, record this information in the data collection table for scenario three. <a href="https://www.jove.com/CDNSource/lm/labs/23/Lab_23_Tables_3_and_4.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Tables 3 and 4</a>
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Add one pipe cleaner of the same length as each of the remaining ones to restore the population to 100. This will likely require more pipe cleaners than were originally cut. Try to use scraps from the class to reduce waste.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Repeat this for three more rounds, alternating which partner holds or grabs each round.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">To perform Scenario 4, gather the varied-length pipe cleaners in their original proportions.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Now repeat the grabbing process, as described in Scenario 3. However, only remove 30 pipe cleaners instead of 50.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Divide the count at the end of each round by two for each phenotype 4 inches or below, rounding fractions down.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Then, record these values in the data recording table for Scenario 4.
</div></li><li data-sub-note-item="1-30"><div class="lab-step">Use the relative proportions of the phenotypes in the final population to restore the population to 100. When restoring the population to 100, it may be necessary to calculate relative proportions of phenotypes.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Repeat this for three more rounds, again alternating holding and grabbing the pipe cleaners.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">For each scenario calculate the survival for each phenotype, that is the proportion of the phenotype surviving.
</div></li><li data-sub-note-item="1-33"><div class="lab-step">Then, calculate the relative fitness, or w, between each round. As reproductive rate does not vary in this simulation, this should always be considered equal, or 1.
</div></li><li data-sub-note-item="1-34"><div class="lab-step">Also, calculate the selection coefficients for each phenotype.
</div></li><li data-sub-note-item="1-35"><div class="lab-step">Now, use the relative frequencies of each phenotype to create histograms with appropriately labeled axes for each scenario showing the population before any selection took place and then alongside these in a different color after four rounds of selection.
</div></li><li data-sub-note-item="1-36"><div class="lab-step">Identify the type of selection taking place in each scenario and label the graphs. 
</div></li><li data-sub-note-item="1-37"><div class="lab-step">Develop a hypothesis to explain your observations. 
</div></li></ul></li></ol>

Natural Selection: Calculating Fitness and Selection - Procedure

自然选择

Simulating Natural Selection
ExpandTo begin, cut the white, black, and gray pipe cleaners into quarters, which should produce a total of 100 pieces that ...

Artificial Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing Artificial Selection in Plants 
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin the experiment, collect five two-week old grown plants from the generation one population that appear healthy. Note: Plants should appear green, not wilted, and not have damaged leaves. Experimental Hypothesis: The experimental hypothesis for this lab might be that the number of lead trichomes varies naturally, but the average number will increase over generations if only plants with the highest number of trichomes are interbred or decrease if those with the least are interbred. Null Hypothesis: The null hypothesis might be that the average number of trichomes will not change over subsequent generations after artificial selection.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Label each plant with a letter from A to E and with both partners&#8217; names.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Record any natural differences you see between your plant samples. Note: All of the plants are the same age, so any observed variation is due to expressed phenotype, not development.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Use a hand lens to count the number of trichomes found on the petiole of the lowest leaf, not including the round, first-grown cotyledons for each plant. Note: It can be helpful to shine a light onto the plant and to hold it against a dark background, such as a lab bench, during the trichome analysis. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Both partners will conduct counts on each plant independently and compare answers. If the counts are different, enlist a third counter or take the average of the two results.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After this, determine the median number of trichomes for the entire class.  <a target="__blank" style="display:block;padding:20px;" href="https://www.jove.com/CDNSource/lm/labs/2/Lab_2_Table_A.xlsx">Click Here to download Table A</a>
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Determine the top and bottom 10% of the plants from the class trichome count. These groups will make up the two artificially selected lines: high hairiness and low hairiness, respectively.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">If more than 10% of the plants have no trichomes, select 10% of those plants randomly for the low hairiness plant line.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Separate the two groups of selected plants and label them clearly.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Move the plants to a well-lit area and keep them well watered until they develop flowers. Note: Flowers typically develop around 14 days.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the flowers are fully developed, use a pollination wand or paintbrush to rub the stigma and anther of one plant&#8217;s flowers for a few seconds.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Using the same wand or brush, rub the inside of each flower of every other plant in the same selection line.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Using a different wand or brush, repeat steps 11 through 12 on the other line. Note: Take care not to cross the lines. Spread the pollen only among the appropriately selected plants.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Return the generation one plants to the growing areas. When the plants reach approximately 1 month old, they should produce mature seed pods.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the plants to dry out once the seed pods turn yellow.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Break the seed pods open and collect the seeds.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Obtain new pots for the generation two seeds.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Fill the pots with moist potting soil about halfway and add a fertilizer pellet to each pot.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Fill the pots with more soil to fill just below the lip.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Make a shallow depression in the soil of each pot with a probe.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Touch a clean toothpick to a gluestick and use the toothpick to transplant the seeds into the soil one by one.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Cover the seeds with a thin layer of soil.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Label the pots with their hairiness line and place them in the designated growing area.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Use a plastic dropper to water the plants as needed to ensure they do not dry out.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">When the plants are two weeks old, collect five plants from each line and label them A to E.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Then, count the trichomes on these plants as previously described (steps 4- 5).
<a target="__blank" style="display:block;padding:20px;" href="https://www.jove.com/CDNSource/lm/labs/2/Lab_2_Table_B.xlsx">Click Here to download Table B</a>
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Data Analysis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Create a frequency graph based on the generation one class data with the trichome count on the x axis and the number of plants on the y axis. Label the mean. 
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, create frequency graphs for both of the selected hairiness lines.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare the frequency graphs for generation one and both hairiness lines.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Take note of phenotypic shifts observed in the selected hairiness lines.
</div></li></ul></li></ol>

Artificial Selection: Visualization in Plants and Data Analysis - Procedure

人工选择

Observing Artificial Selection in Plants 
ExpandTo begin the experiment, collect five two-week old grown plants from the generation one population that ...