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.

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。 1. 在 C1498细胞系的体外表征<ol><li> 在 C1498细胞的体外培养<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 7个细胞/ ml和分配10 6个细胞（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（ 例如 ，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10个结果。 </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。 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 7个细胞/ ml和分发10 6个细胞（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10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 6rvested 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 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> 2. 在体内发育和急性白血病的表征注: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（ 即，在无菌环境中）。当他们周5和6岁之间的小鼠注射。 <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 7个细胞/ 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 6个细胞）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（ 例如 ，立毛，从组隔离，并减少或在笼不运动）的迹象的行为。 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。 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 6个细胞。 </li><li>至10 6个骨髓细胞，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10。 </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。 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10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 5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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The authors declare that they have no competing financial interests.

在以前的研究中，C1498细胞系被描述为急性粒5，6粒单核细胞或NKT 7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。 当我们在体外培养的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6,7之前已经描述几个细胞表面造血标记。与我们的结果一致，拉贝勒等人没有使用流动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 7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。 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 +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。 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 + 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 +）。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 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（ 例如 ，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（ 例如 ，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 +）和单核细胞（CD115 +）细胞阳性和阴性对照载玻片进行测试。 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11,12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。 这是秀N， 使用体外实验，即C1498细胞通过分泌可溶性因子13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14。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15。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16。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。

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1。 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。 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 B）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（ 例如 ，脾和淋巴结）和非造血器官（ 例如 ，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2-4。然而，这种小鼠模型报道诱发要么粒2,5或6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7。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。 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在体外培养的细胞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。 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8,9。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。

<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

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，我们在体外表征C1498细胞和用于确定在体内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在诱导白血病病体内。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（ 例如 ，骨髓和脾）和非淋巴组织（ 例如 ，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。

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，以确定其体外造血谱系和成熟阶段（ 图1）。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。 为了表征体外 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（ 表1）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（ 图2A和B）。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（ 表1）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 b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）。 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（ 图3A）。大多数细胞还沾着积极为α萘丁酯酶（ 图3B，左图 ），其中一些染色的色酚AS-D氯乙酸酯酶（黑色箭头）（ 图3B，右图 ）。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（ 图3C ）。钍美国，C1498细胞系是由monoblasts和原粒细胞。 的C1498细胞（CD45.2 +）然后静脉注射到CD45.1 +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（ 图4A）。他们还保存他们的单核细胞和粒表型（ 图4B和C），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。 到Determine以下白血病细胞浸润，CD45.2 + 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）（ 图5A至C）。 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（ 图6A）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11（ 图6B）的。 间急性单核细胞白血病12的另一特点，C1498注射小鼠带有肿肝脏（肝），肺和脾（脾肿大）（ 图7A）。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（ 图7B）CD45.2 + 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（ 图7C）。 薄页="1"&gt; <img alt="图1" src="/files/ftp_upload/54270/54270fig1.jpg" /> 首先确定图1中的协议设置为在体外培养的C1498细胞系和在急性白血病的体内描述表征。造血谱系和组织培养C1498细胞的分化阶段的示意图 。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MGG: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> <html>广告/ 54270 / 54270fig2.jpg"/&gt; 图2. C1498细胞表型分析在体外培养。代表流式细胞仪散点图和细胞表面（A）和细胞内（B）的直方图后，C1498-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> <html> ntent"FO:保together.within页="1"&gt; <img alt="图3" src="/files/ftp_upload/54270/54270fig3.jpg" /> 图3.功能和培养C1498细胞的形态学特征 。从培养物收获并离心在玻片上进行显微镜C1498细胞。（A）中 ，使用免疫荧光进行用于髓过氧化物酶表达染色。（B）中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。（c）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。 <img alt="图4" src="/files/ftp_upload/54270/54270fig4.jpg" /> 图4.骨髓形貌在PBS-和C1498注射小鼠骨髓细胞从PBS-和C1498细胞注射的小鼠中分离，离心到幻灯片显微镜（A）可以-Grünwald的吉姆萨（MGG）染色。（B ）α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（C）的萘酚。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> <img alt="图5" src="/files/ftp_upload/54270/54270fig5.jpg" /> 图5.髓质种群PBS-和定量分析C1498注射小鼠骨髓细胞从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。（A）中的B细胞亚群包括CD19 + B220 +细胞从原B细胞阶段到成熟B淋巴细胞（B）的粒细胞中的CD3 -和CD11b + Ly6G +谱系，包括前体的。第二未成熟和成熟粒细胞（C）中的单核细胞亚群被定义为CD3 - CD115 +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t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> <img alt="图6" src="/files/ftp_upload/54270/54270fig6.jpg" /> 在PBS-和单核细胞亚群图6.血液分析C1498注射小鼠 。代表流式细胞仪已分别定义为CD3 +和B220 +细胞在PBS-C1498和（一）T，B淋巴细胞百分比，散点图细胞注入以及CD115 +的Ly6C 高细胞-小鼠（B）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 +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 +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> <img alt="图7" src="/files/ftp_upload/54270/54270fig7.jpg" /> 在白血病和对照组小鼠脾种群图7.估计。 （A）的肝，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。（B）柱状图表示差异白血病细胞频率erent器官（℃）后，对CD45.2 +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 + B220 + ，CD3 -细胞CD11b + Ly6G +，CD3 -细胞CD11b +的Ly6C -和CD3 -细胞CD11b +的Ly6C 高细胞。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 * P &lt;0.05; **，p值= 0.0033，非配对t-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> <table fo:keep-together.within-page="1"><tbodY&gt; <tr><td> 电池类型 </td><td> 膜或细胞内分子 </td></tr><tr><td></td><td></td></tr><tr><td> 前体和成熟细胞 </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> 先觉 </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> 造血干细胞 </td><td> CD117 +的Sca-1 + CD150 +（Lin-的CD34-） </td></tr></tbody></table> 表1.造血细胞谱系和分化的标志物。 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。

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

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。

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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19.9K 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

Balmer Series

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit around the nucleus. Importantly, an atom cannot discharge energy while its electrons are in stationary states. An electron can only emit energy by changing energy states. To change energy states, an electron must move from one orbit to another by either absorbing or emitting energy. This change can only occur if the absorbed or emitted energy equals the difference between the two states. Electrons cannot exist between orbits.
The quantum number, n, is used to label the different energy states. The lowest energy state is the ground state, which is n equal to one. The excited states are labeled n equal to 2, 3, 4, and so on. When an electron at the ground state absorbs a photon whose energy equals the difference between the ground and second state, the electron becomes excited and transitions from the ground state to the n= 2 excited state. If the energy of the photon equals the difference between the ground and third state, the electron moves to the n=3 state.
According to Bohr&#8217;s model, the potential energy of an electron in the nth level can be calculated using the following equation:
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_1.jpg" />
where En is the potential energy, R is the Rydberg constant (1.0974 &#215; 107 m-1), h is Planck&#39;s constant (6.62607004 &#215; 10-34 m2&#183;kg/s), and c is the speed of light (~ 3 &#215; 108 m/s). Electrons can also spontaneously return to the ground state or any other lower excited state. When this happens, the excess energy is released in the form of an emitted photon. The energy of the photon is equal to the energy difference between the higher and lower energy states. That energy corresponds to wavelengths of light. Since each type of atom has different energy levels, the light emitted from each transition varies for each atom. For a sample of mixed molecules, the emitted light contains a range of wavelengths in what is called a continuous spectrum. For a sample containing atoms of a single element, the emitted light contains only certain wavelengths, which can be viewed as discrete lines once separated by a prism.
<h4>The Hydrogen Atom</h4>
Looking specifically at the hydrogen atom, the excitation of its electrons requires the absorption of sufficient energy to split the bond in the diatomic molecule H2. Since more energy is used to split the molecule than needed, the electrons in the hydrogen atom absorb the excess energy and are excited to a higher energy level. When the electrons spontaneously return to a lower energy level, light is emitted, which corresponds to the energy difference between the excited level and lower level.
When discussing the emission of energy, the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. The wavelengths of emitted light ultimately depend on the energy difference between the two levels.
In a pure sample of hydrogen gas, the emission spectrum appears as distinct lines of discrete wavelengths that are specific to the element hydrogen. Some of these lines are in the visible range of the electromagnetic spectrum, while some are in the ultraviolet or infrared range.
<h4>The Balmer Series</h4>
The series of visible lines in the hydrogen atom spectrum are named the Balmer series. This series of spectral emission lines occur when the electron transitions from a high-energy level to the lower energy level of n=2. Johann Balmer observed these spectral lines at 410.2 nm, 434.1 nm, 486.1 nm, and 656.3 nm, which correspond to transitions from the n=6, n=5, n=4, and n=3 energy levels to the n=2 level, respectively.
Balmer was able to relate these wavelengths of emitted light using the Balmer formula.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_2.jpg" />
Here, &#955; is the observed wavelength, C is a constant (364.50682 nm), n is the lower energy level with a value of 2, and m is the higher energy level, which has a value greater than 3. This observation was then refined by Johannes Rydberg, where R is the Rydberg constant.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_3.jpg" />
Remember that this equation describes emitted light, so the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. In the case of the Balmer series, nf is equal to 2. This equation was combined with the Bohr model to calculate the energy needed to move an electron between its initial and final energy levels, &#916;E.
<img src="https://www.jove.com/CDNSource/lm/labs/54/54_Concepts_4.jpg" />
Later, other spectral series for the hydrogen atom were discovered. For example, the Lyman series contains emission lines with energies in the ultraviolet region.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silderberg, M.S. (2009). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill.</li>
</ol>

The Hydrogen Atom: Bohr Model and Balmer Series - Concept

Balmer 系列

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit ...

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化学

Genetics of Organisms

<h4>Mendelian Genetics</h4>
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this process as changes in the frequency of alleles at individual genetic loci. This simple representation of how evolution occurs dates to Gregor Mendel&#8217;s analysis of trait inheritance patterns in pea plants, first presented in 1865. Mendel determined, using rigorous collection of data, that noticeable traits are controlled by two alleles of each gene (he called them factors), one from each parent. In addition, he concluded that some alleles are dominant to others (deemed recessive). Interestingly, Mendel&#8217;s data was not appreciated until 1900, probably in part because his approach to the study of inheritance was so divergent from his predecessors. Even after his brilliant experiments were rediscovered, it took 30 additional years for Mendelian inheritance to become integrated with evolutionary theory and to gain universal acceptance.
Why was such a seemingly clear and straightforward process dormant for so long and then the center of controversy for decades? One key to the controversy is that we rarely see the clear patterns of inheritance described by Mendel. Animal and plant breeders most often see evidence for what was described as blending inheritance, or offspring whose phenotypes are intermediate between those of their parents, rather than the discrete segregation of traits into well-defined categories, like smooth versus wrinkled peas or red versus white flowers. Mendelian inheritance thus did not align well with practical experience and it took decades to reconcile the two. Additionally, there are now known to be modes of inheritance that, at first glance, appear to violate Mendel&#8217;s laws. Deeper investigations into genetics using molecular biology have effectively characterized these novel modes of inheritance. The new information does not invalidate Mendel&#8217;s findings, however, it simply enhances his model.
<h4>Phenomena that Violate Mendelian Rules</h4>
There are three main phenomena: epistasis, pleiotropy, and sex-linkage that appear to violate the basic dominance and recessivity rules of Mendelian inheritance. Epistasis occurs when two or more genes or alleles interact to affect a phenotype. A function of one gene product may be required to allow another gene to be expressed or to function normally. On the other hand, pleiotropy occurs when one gene controls the expression of multiple phenotypes in an individual. For example, a large proportion of cats with white fur and blue eyes are deaf 1. Pleiotropy is often antagonistic, meaning that the same gene causes beneficial changes in one facet of an individual&#8217;s phenotype while at the same time causing damaging changes in some other aspect of an individual&#8217;s phenotype. Antagonistic pleiotropy is seen as a tradeoff or constraint on evolution, as proposed by George C. Williams. For example, the age specific decline in performance, or senescence, is thought to be a pleiotropic trait controlled by the same genes that increase early-life fecundity2,3.
<h4>Sex Linkage</h4>
Sex linkage is another phenomenon discovered as a counter-example to basic Mendelian inheritance. In many organisms, pairs of sex chromosomes determine gender. In humans and many mammals, XX individuals are female while XY individuals are male. Male offspring inherit an X from their mother and a Y from their father; females inherit an X from their mother and an X from their father. In most species, the ratio of males to females at birth under &#8220;normal&#8221; environmental conditions is 1:1. Y-chromosomes undergo little recombination with X chromosomes during meiosis, due to the fact that, over evolutionary time, they have lost a substantial number of genes. Interestingly, there are only 16 genes shared by the human X and Y chromosomes4.
For X-linked traits (traits encoded by genes on the X chromosome) females can be homozygous (having two copies of the same allele) or heterozygous (having two different alleles). If the trait is recessive, females will only show the phenotype for the trait if they are homozygous. Heterozygous females &#8220;carry&#8221; the recessive allele, but do not express the phenotype of that allele. Statistically speaking, half of her sons will inherit this recessive allele and all will express the phenotype because males have only one X chromosome.
A commonly used example of an X-linked phenotype is hemophilia, a disease that prevents the synthesis of blood clotting proteins. If a female is heterozygous (is a carrier, genotype XH Xh) mates with a normal male (genotype XH Y), 50% of the sons will be affected. If the female was instead homozygous for the trait (genotype Xh Xh ), none of her daughters would be hemophiliacs but 100% of her sons would be. The differential phenotypic ratios seen in sons and daughters appears to violate Mendel&#8217;s laws, yet Mendel&#8217;s first law still applies; each offspring has a 50% chance of inheriting an X-chromosome carrying the hemophilia allele from a heterozygous mother5.
Like humans, the fruit fly Drosophila melanogaster has an XY-sex determination system. D. melanogaster make excellent laboratory organisms because they are easy to keep, breed, and manipulate. Wild-type flies have what is considered normal body morphology and red eyes. Many lines are readily available for purchase, including some with mutations resulting in flies without eyes, with eyes of varying color, or missing wings. These organisms can be used to study or demonstrate both Mendelian and non-Mendelian inheritance patterns. In fact, 1933 Nobel Prize winner, Thomas Hunt Morgan, first noticed that some genes are sex-linked using this system6.
<h4>References</h4>
<ol class="references">
	<li>Strain, George M., &#39; Cat Breeds With Congenital Deafness,&#39; [Online]. Available: https://www.lsu.edu/deafness/catbreeds.htm. [Accessed 29 August 2018].</li>
	<li>W. GC., &#39;Pleiotropy, natural selection, and the evolution of senescence.,&#39; Evolution, vol. 11, p. 398&#8211;411, 1957.</li>
	<li>T. &#38; S. P. S. Flatt, &#39;Integrating Evolutionary and Molecular Genetics of Aging,&#39; Biochimica et Biophysica Acta, vol. 1790, no. 10, p. 951&#8211;962, 2009.</li>
	<li>D. Bachtrog, &#39;Y chromosome evolution: emerging insights into processes of Y chromosome degeneration,&#39; Nature Reviews. Genetics, Vols. 14(2), 113&#8211;124, 2013.</li>
	<li>US National Library of Medicine, &#39;Hemophilia,&#39; Genetics Home Reference, [Online]. Available: https://ghr.nlm.nih.gov/condition/hemophilia. [Accessed 31 August 2018].</li>
	<li>&#39;The Nobel Prize in Physiology or Medicine 1933,&#39; [Online]. Available: https://www.nobelprize.org/prizes/medicine/1933/summary/. [Accessed 31 August 2018].</li>
</ol>

Genetics of Organisms: REF and WEF Fly Genetics - Concept

生物体的遗传学

Mendelian Genetics
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this ...

生物学

减数分裂

Animal Behavior

<h4>Drosophila as a Model Organism</h4>
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly recognized as a fruit-loving pest. There are multiple reasons that make D. melanogaster an excellent experimental organism: their roughly two-week generation time allows studying multiple generations, they are easily maintained in very small tubes, and their sexual dimorphism enables investigators to easily distinguish between males that have black coloration towards the posterior and females that have banded abdomens.
Thomas Hunt Morgan and his students were among the first to make use of Drosophila, in the early 20th century. In 1933, Morgan received a Nobel Prize for his discovery of the role of chromosomes in heredity, which was founded on his observations of mutant Drosophila. Morgan continued his studies by generating new mutants with radiation. The presence of multiple mutants with discrete effects on the appearance of the fly enabled him to look at patterns of multifactor inheritance. A key aspect of his early work was the discovery of apparent violations of Mendel&#8217;s second law, which states that genes segregate independently of one another. Morgan&#8217;s observations indicated that genes will not segregate independently if they are joined together on the same chromosome. Furthermore, it became possible to determine gene locations on a chromosome relative to each other through analyses of how often they recombined thus triggering gene mapping studies to identify the location of a gene and distances between genes. Consequently, associates of Morgan&#8217;s lab received two additional Nobel Prizes for other studies of Drosophila genetics. Morgan&#8217;s success and the accumulating information about Drosophila inspired others to adopt them as experimental organisms, which added to our knowledge of them and progressively increased their value as a model organism. Other scientists have developed many mutations. Over 27,000 unique Drosophila lines are now maintained as stock cultures at the Drosophila stock centers and are readily available to laboratories that use them as tools for studying diverse aspects of biology, including behavior1.
<h4>Drosophila Ethology</h4>
Animal behavior, or ethology, is the study of how animals interact with each other and their environments. Although it is a relatively young biological field, ethology has important implications since an organism&#8217;s survival depends on how it behaves with regard to possible mates, food sources, and natural obstacles. Studying these organisms helps scientists to understand the contexts of these behaviors and the underlying mechanisms as to how they have evolved. Thus, a goal of the study of animal behavior is to find a genetic basis for the behaviors exhibited by individuals or groups of individuals. For example, kin selection, first proposed by William Hamilton, suggests that individuals will behave altruistically when making a sacrifice for a family member rather than a stranger2. This is because related individuals share more genes than unrelated ones, and the future reproduction by their relatives contributes in a way to their own fitness, or reproductive success. Hence, observing how organisms like Drosophila interact with their environment can allow us to understand how these behaviors are hard-wired. Additionally, these behaviors provide insight into how these organisms have adapted to cope with their environment and survive.
Directional behaviors are commonly studied to understand the basis of how and why an organism moves in its environment. Kinesis and taxis are two forms of directional behaviors. Kinesis involves stimulus-induced movements in random directions, such as the random movement of flies when chased away. On the other hand, taxis involves stimulus-induced movement towards a specific direction, such as moths flying towards a lightbulb. Taxis behaviors are positive if the animal moves towards the stimulus and negative if the animal moves away from the stimulus. Furthermore, taxis behaviors are named based on the specific stimulus that induces them. For example, geotaxis is a response to gravity, where positive geotaxis means that the organism moves with gravity. Phototaxis is movement in response to light, such as positive phototaxis of moths towards a lightbulb. Similarly, chemotaxis is attraction to or avoidance of an airborne chemical cue, such as the negative chemotaxis of many predators, including humans, in response to the smell of skunk spray. Furthermore, there are many other types of taxes that are studied by scientists, including aerotaxis, barotaxis, hydrotaxis, and magnetotaxis, that are movements in response to oxygen, pressure, water, and magnetic fields, respectively.
<h4>Study of Drosophila taxis behavior</h4>
A common and simple method to study Drosophila taxis behavior is to use a choice chamber, which allows the observation of a directional response of the fly to a particular stimulus, such as gravity, light, or a chemical. Drosophila are inserted in the center of the choice chamber and then allowed to wander freely about the chamber, which is designed so that alternative stimuli can be presented at opposing sides. After a period of wandering, the number of flies is counted on either side of the choice chamber. The number of flies on each side of the chamber is then analyzed with the Chi Square test to gauge whether or not there is a preference for a stimulus.
Comparison of directional behavior of mutant and wild-type Drosophila allows researchers to determine the role of the mutated gene in that specific behavior. Moreover, scientists are also trying to understand how animals decide what to do in the presence of combinations of stimuli, including conflicting stimuli3. Together, these studies can help predict how organisms will react to certain stimuli or changes in environmental factors.
<h4>References</h4>
<ol class="references">
	<li>Cook KR, Parks AL, Jacobus LM, Kaufman TC, Matthews K. New research resources at the Bloomington Drosophila Stock Center. Fly. 2010, Vol. 4, 1: 88-91.</li>
	<li>WD, Hamilton. Altruism and related phenomena, mainly in social insects. Ann Rev Ecol Systematics. 1972, Vol. 3, 193-202.</li>
	<li>Gepner R, Skanata MM, Bernat NM, Kaplow M, Gershow M. Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration. eLife. 2015, Vol. 4, e06229.</li>
</ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Concept

动物行为

Drosophila as a Model Organism
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly ...

行为

Hardy-Weinberg and Genetic Drift

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are therefore very difficult to measure. In general, studying phenomena like this requires the use of mathematical models which are built using parameters that can be conveniently measured. These models are then used to make predictions about how changes to the system might affect the outcome. 
For example, changes to the frequency of genetic alleles at individual loci in species are often observed over long time periods, but are not generally observable over short time periods. The use of computer models allows researchers to predict changes within a species&#8217; gene pool using observations that have already been gathered, and allow for simulations of a potentially unlimited number of generations of the population. This would certainly not be possible within a human lifetime.
<h4>The Hardy-Weinberg Equilibrium Principle</h4>
One equation used to model populations is the Hardy-Weinberg equilibrium equation. It was formulated independently in 1908 by both G. H. Hardy and Wilhelm Weinberg1,2. The simple equation describes the expected allele frequency of a population that is not evolving. Because most real-life populations are evolving in response to the forces of natural selection, this formula acts as a useful null hypothesis. It is used to test whether or not different types of selection are, or are not, occurring. If measured allele frequencies differ from those that were predicted using the Hardy-Weinberg equation, then the gene in question is undergoing evolutionary change.
The Hardy-Weinberg equation can be represented as p2 + 2pq + q2 = 1 where p represents the frequency of the dominant allele and q represents the frequency of the recessive allele for the gene in question. Each expression in the equation represents the predicted frequency of one of the three possible diploid genotypes. Homozygous dominant frequency is represented by p2, homozygous recessive by q2, and heterozygous by 2pq. If these frequencies are summed, it adds up to 1, which makes sense since the full population had been divided into the three available categories.
It is also possible to describe the population, or gene pool, in terms of the two alleles, without regard to how they are packaged into diploid individuals. This equation is represented as p + q = 1, that is, the frequencies of the dominant and recessive alleles must add up to 1 within the population. Again, this makes sense, since the model includes just two allele possibilities. Notice that the frequency of genotypes in the population is just the quadratic expansion of the frequency of alleles in the population because (p + q)2 = p2 + 2pq + q2.
The Hardy-Weinberg equation, like most other models, requires a series of assumptions. Usefully, if the real-life data differs from the predicted data, it is possible to hypothesize which of the starting assumptions was false. The assumptions of the Hardy-Weinberg equilibrium equations are: 1) the population is very large, 2) the population is closed, meaning that there are no individuals immigrating into or emigrating out of the population, 3) there are no mutations occurring on the gene in question, 4) individuals within the population are mating at random&#8212;individuals are not choosing their mates, 5) natural selection is not occurring. Again, keep in mind that the Hardy-Weinberg equilibrium equation is a null hypothesis. Frequently, real populations violate one or more of these assumptions. The equation is used to determine if and how evolution is happening.
<h4>Genetic Drift</h4>
In a relatively small population, a condition that violates the first Hardy-Weinberg assumption, it is possible for allele frequencies to have resulted from chance. This phenomenon is referred to as genetic drift. One version of this is referred to as the founder effect. If a small number of individuals move to an isolated location and start a new population, the specific genetics of those particular individuals will shape the future generations. This new small gene pool may have the same allele frequency as the original, but it is also possible, even likely, that it does not. Say the original population included 50% dominant alleles and 50% recessive. In the extreme, if all of the individuals in a migrating founder group are homozygous recessive, then the dominant allele has been lost completely and the recessive is now at 100%. A similar phenomenon, called the bottleneck effect, can occur if a population sustains a large drop in numbers due to a natural disaster, human intervention, or disease.
Importantly, genetic drift represents a type of evolution that is not necessarily adaptive. It does not specifically select for traits that are fit for the environment. Nonetheless, it is an important evolutionary force in shaping populations with a small number of individuals and will cause deviations from Hardy-Weinberg predictions. In populations that are larger, deviations from the predictions are more likely to mean that the population in question is undergoing evolution through natural selection. Under these circumstances, further consideration of the mechanisms causing this evolution can be explored.
Modern applications of the Hardy-Weinberg model include analysis of human and animal populations in terms of how their immune systems relate to their susceptibility to infectious disease3,4. Many research groups are cataloging genes which encode specific immune system molecules, such as CCR5 and the major histocompatibility complex (MHC, known in humans as the human leukocyte antigen (HLA), and correlating this information to epidemiological studies regarding disease and its progression3,5-6. From comparing these two types of data sets, patterns are emerging showing that some individuals are genetically resistant to particular infections while others are more likely to succumb to disease4. Hardy and Weinberg&#8217;s model has been essential to the analysis of this data and has contributed to our understanding of how infections have shaped evolution.
<h4>References</h4>
<ol class="references">
	<li>Hardy, G. H. (Jul 1908). 'Mendelian Proportions in a Mixed Population' (PDF). Science. 28 (706): 49&#8211;50. doi:10.1126/science.28.706.49. ISSN 0036-8075. PMID 17779291.</li>
	<li>Weinberg, W. (1908). '&#220;ber den Nachweis der Vererbung beim Menschen'. Jahreshefte des Vereins f&#252;r vaterl&#228;ndische Naturkunde in W&#252;rttemberg. 64: 368&#8211;382.</li>
	<li>Eguchi S, Matsuura M. Testing the Hardy-Weinberg equilibrium in the HLA system. Biometrics. 1990 Jun;46(2):415-26. PubMed PMID: 2364131.</li>
	<li>Phillips KP, Cable J, Mohammed RS, et al. Immunogenetic novelty confers a selective advantage in host-pathogen coevolution. Proc Natl Acad Sci U S A. 2018;115(7):1552-1557.</li>
	<li>Lim JK, Glass WG, McDermott DH, Murphy PM CCR5: no longer a 'good for nothing' gene--chemokine control of West Nile virus infection. Trends Immunol. 2006 Jul; 27(7):308-12.</li>
	<li><a target="__blank" href="https://hla-net.eu/tools/frequency-estimation/">https://hla-net.eu/tools/frequency-estimation/</a></li>
	<li>Santiago Rodriguez, Tom R. Gaunt and Ian N. M. Day. Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies. American Journal of Epidemiology Advance Access published on January 6, 2009, DOI 10.1093/aje/kwn359. http://www.oege.org/software/hwe-mr-calc.html</li>
</ol>
<h4>Further Reading</h4>
Andrews, C. (2010) The Hardy-Weinberg Principle. Nature Education Knowledge 3(10):65 https://www.nature.com/scitable/knowledge/library/the-hardy-weinberg-principle-13235724

Hardy-Weinberg Equilibrium Principle and Genetic Drift - Concept

Hardy-Weinberg 和遗传漂变

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are ...

进化

Evolutionary Relationships

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th 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.
<h4>Types of Dendrogram</h4>
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 group1.
<h4>Understanding Evolutionary Relationships using Genetic Data</h4>
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 ancestor1. 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.
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.
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) database2. 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 query 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.
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, Drosophila melanogaster, or the mouse3. 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.
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 Cftr knockout mouse and another modeling atherosclerosis, called the Apoe knockout3. 
 
<h4>References:</h4>
<ol class="references">
	<li>Annenberg Learner. Online Textbook: Unit 3 Evolution and Phylogenetics. Rediscovering Biology. [Online] 2017. [Cited: August 23, 2018.] https://www.learner.org/courses/biology/textbook/compev/compev_3.html.</li>
	<li>National Center for Biotechnology Information, U.S. National Library of Medicine. BLAST: Basic Local Alignment Search Tool. [Online] https://blast.ncbi.nlm.nih.gov/Blast.cgi.</li>
	<li>National Human Genome Research Institute. Background on Mouse as a Model Organism. National Human Genome Research Institute. [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 ...

Animal Diversity

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular eukaryotes that move, consume organic matter, and reproduce sexually. Although these attributes are shared, species within this kingdom are also extremely diverse. This diversity is due to adaptation of each species to a different niche. The niche of a species includes the area, function, and interrelationship of that species with other biotic and abiotic factors in its environment. Niche specialization through evolutionary adaptation allows species to survive and reproduce effectively in their environment and reduces competition among species within the same habitat1-2.
<h4>Classification</h4>
Diversity in Kingdom Animalia has led to the classification of 36 distinct phyla, based on their evolutionary lineage. Seven of these phyla will be discussed, including Porifera, Cnidaria, Platyhelminthes, Annelida, Mollusca, Arthropoda, and Chordata. The first, phylum Porifera, is the earliest, simplest, most ancestral phylum. It includes multicellular, asymmetrical filter feeding sponges that lack distinct tissue layers. Interestingly, sponges can regrow after being broken apart to the level of a single cell. Next, increasing in complexity, the phylum Cnidaria includes jellyfish and coral. Members of this phylum are radially symmetric and diploblastic (possessing two tissue layers). These tissue layers are called the endoderm, which makes up the inner layer, and the ectoderm, which forms the outer layer. Organisms in phylum Cnidaria contain a simple gut with a single opening through which food enters and waste is excreted. Both Porifera and Cnidaria include organisms that are largely sedentary as adults. However, the remaining phyla have evolved adaptations that allow movement and more sophisticated behavior.
Following Cnidaria, there are two phyla of worms, termed Platyhelminthes and Annelida. Worms from both of these phyla breathe by exchanging gases across their skin. As a result, most worms have a large surface area to volume ratio and live in moist or aquatic environments. The skin of these organisms must remain moist to facilitate effective diffusion of gases. Another common characteristic of worms is their bilateral symmetry. This means that an organism can be symmetrically divided in half along a plane. Bilateral symmetry is seen not only in worms, but also in every phyla from this evolutionary point forward. Such symmetry is important to allow directed movement and to concentrate sensory organs near the anterior tip, or head, where they interact with their surroundings. Worms and later phyla also contain an additional tissue layer called the mesoderm that lies between the ectoderm and endoderm. As a result, these organisms are considered &#8220;triploblastic.&#8221;
Of the two phyla of worms, Platyhelminthes is the more ancient and simple. These include flatworms and trematodes, which may be free-living or parasitic. Like Cnidarians, they possess a simple gut with a single opening. Phylum Annelida is more complex, including earthworms, leaches, and polychaeta. These organisms contain a body cavity, called the coelom, between the gut and body wall. They are also distinct due to the segmentation of their bodies. Division of an organism into segments was an important evolutionary step, allowing adaptation of each segment to specialized forms and functions. Major evolutionary advances in this phyla include the development of anterior segments with appendages specialized for grasping and processing food as well as posterior segments with appendages specialized for locomotion.
The next phylum, Mollusca, contains highly morphologically diverse organisms, including octopuses, snails, slugs, and clams. There are few defining features for this group. However, all mollusks possess a mantle, which acts as a protective cover for the respiratory organs, digestive tract, and reproductive structures. Next, the most diverse group of organisms is phylum Arthropoda. This phyla alone makes up approximately three quarters of all living and extinct species known today, including crustaceans, insects, and arachnids. All members of this phylum have a hard, outer coating made of chitin, called an exoskeleton. This exoskeleton is periodically shed for a new, larger one to take its place. Like Annelida, arthropods are segmented. However, instead of multiple repeating sections, arthropods are broken up into three distinct segments called the head, thorax, and abdomen. Most have specialized sensing structures attached to the head called antennae.
Lastly, phylum Chordata includes all animals with a backbone and a few without. Two defining features that unite this group are the presence of a nerve chord and a notochord. The nerve cord is a major part of the chordate nervous system and the notochord is a cartilaginous structure that runs between the nerve cord and the digestive tract. Phylum Chordata includes tunicates (which lose their nerve cord and notochord after metamorphosis from the larval to adult life stage), fish, amphibians, birds, reptiles, and mammals (including humans)3.
<h4>Form and Function</h4>
The vast diversity in body structure, habitats, and behavior observed across all 36 phyla within Kingdom Animalia is truly astounding. To understand these differences better, it is important to consider the connection between form and function. &#8220;Form&#8221; simply means the shape, size, and substance of a structure or organism while &#8220;function&#8221; stands for the way in which that structure is used. Accordingly, the function of a structure can be predicted based on the form observed, or vice versa. For example, a bird specialized to crack hard nuts would be predicted to have a sturdy, sharp beak while a bird that eats fruit may have a smaller, more rounded beak.
Importantly, the form of an organism is constrained by its physiological requirements. For instance, all animals must consume and digest organic matter and exchange gas with their environment. As a result, any adaptations to an animal&#8217;s form must continue to facilitate these processes efficiently. For example, in smaller animals, diffusion of gases across the skin is possible and often useful. However, as the size of an animal increases, its surface area to volume ratio decreases until diffusion across the skin can no longer provide sufficient gas exchange. Such larger animals have developed complex systems to deal with gas exchange, such as the lungs of a whale or the gills of a fish. As a result, the morphology of gas exchange systems depends on both the size of an organism and its environment.
Similarly, it generally follows with other types of adaptations that form follows function, constrained only by the physiological requirement of the organism. However, there are cases in which form does not follow function. Vestigial structures are those that served a purpose in an organism&#8217;s ancestor but are no longer useful in their current form. Such structures have little to no impact on the fitness of an individual, and are thus not evolutionarily selected for or against within the population. As a result, some structures persist with limited functional capacity. For example, the human appendix, a small pocket at the end of the large intestine, is an evolutionary remnant from our ancestors. While this structure has been hypothesized to store beneficial gut bacteria, removal of the appendix is possible with no deleterious effects.
<h4>Convergent Evolution</h4>
Among adaptations seen in Kingdom Animalia, many have developed multiple times throughout evolutionary history. Consider the wings of a bat and those of a bird as an example. These structures are not a result of evolutionary relatedness, but have instead arisen from convergent evolution4. Convergent evolution occurs when two organisms independently evolve similar traits due to similar environmental pressures or niches. The wings of bats, birds, and insets have all developed independently and are said to be &#8220;convergent traits.&#8221; Clearly, such traits are quite beneficial for the niches in which they arise.
<h4>References</h4>
<ol class="references">
	<li>Hollins, J., et al. (2018). 'A physiological perspective on fisheries-induced evolution.' Evol Appl 11(5): 561-576.</li>
	<li>Leong, M., et al. (2017). 'The Habitats Humans Provide: Factors affecting the diversity and composition of arthropods in houses.' Sci Rep 7(1): 15347.</li>
	<li>Wray, G. A. (2015). 'Molecular clocks and the early evolution of metazoan nervous systems.' Philos Trans R Soc Lond B Biol Sci 370(1684).</li>
	<li>Foll, M., et al. (2014). 'Widespread signals of convergent adaptation to high altitude in Asia and America.' Am J Hum Genet 95(4): 394-407.</li>
</ol>

Animal Diversity: Form and Function of Crickets and Crayfish - Concept

动物多样性

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular ...

Microbial and Fungal Diversity

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this reason, it is important to understand and distinguish between individual species of these groups. As you will recall, biological taxonomists group organisms based on their phylogenetic relatedness. The three domains of life, Bacteria, Archaea, and Eukaryota segregate life into three distinct groupings. While all bacteria fall within the domain Bacteria, fungi are found in the domain Eukaryota (Kingdom Fungi). In addition to Archaea, domain Bacteria exclusively includes single-celled prokaryotes, meaning their cells lack both a nuclear membrane and membrane-bound organelles. In contrast, fungi of domain Eukaryota have membrane-bound organelles and include both unicellular and multicellular organisms.
<h4>Bacteria</h4>
Bacteria are found nearly everywhere on Earth and are highly adapted to the environments in which they grow. As a result, this domain includes a staggering amount of diversity. The impact of these organisms on human health is equally diverse. For example, species like Streptococcus thermophilus can be used to produce fermented foods like yogurt, while spores of the bacteria Bacillus anthracis are responsible for the potentially deadly disease anthrax.
To aid in the identification of such organism, microbiologist have developed a myriad of tests to characterize both individual cells and entire colonies of bacteria1. One of the most common methods for identifying bacteria is by Gram staining. This test uses colored dyes to determine the composition of the bacteria&#8217;s cell wall. Gram-positive bacteria, which bear a thick cell wall made up of a protein called peptidoglycan, retain a dye called crystal violet and thus appear purple. In contrast, gram-negative bacteria have a thinner cell wall without peptidoglycan and do not retain the crystal violet. Instead, these cells appear pink due to a counter stain called safranin. The use of Gram staining often serves as a preliminary step in the identification of a bacterial species. Further classification requires the identification of additional characteristics. This can include the shape of individual bacteria, which may be either cocci (round), spirillum (spiral), or bacilli (rod-shaped). Additionally, the color, shape, sheen, texture, and even smell of bacterial colonies can be used to distinguish between species.
<h4>Fungi</h4>
Fungi of domain Eukaryota are also highly ecologically diverse and can range in size from microscopic yeast to the largest organism on earth, the honey fungus (Armillaria ostoyae). All fungi are heterotrophic, meaning that they must acquire their food from the environment. These organisms also exhibit diversity in their specific activities, niches, and potential effects on human health. For example, some fungi are consumed or used to produce food by humans while others can cause severe or even deadly infections2.
Within kingdom Fungi, two distinct taxonomic divisions exist based on the reproductive strategy of the species. First, Ascomycota, known as the sac fungi, have a reproductive structure called an ascus. Together, many asci form the ascocarp, or &#8220;fruiting body&#8221; of the fungi. These fungi can reproduce sexually or asexually by budding. Ascomycota consist of some economically important species like the yeast used in brewing beer and baking bread, and some species popular in cuisine, such as morels and truffles. The second group, Basidiomycota, includes organisms commonly known as &#8220;club fungi&#8221; due to the presence of a large club-shaped reproductive structure called the basidium. In contrast to Ascomycota, most Basidomucota reproduce exclusively by sexual reproduction. This occurs when two haploid mycelia join to form a basidiocarp. On a button mushroom, this is the cap structure. On the underside of the basidiocarp, reproductive structures called gills lined with structures called basidia produce haploid nuclei that eventually form basidiospores. Basidiospores are then dispersed by the wind. This taxonomic division includes many commonly eaten button mushrooms, as well as wild puff mushrooms.
<h4>Importance of Microbiology</h4>
Overall, it is clear that the diverse functions and properties of microorganisms make their identification extremely important for human health and well-being. While a vast number of species of fungi and bacteria have been identified to date, it is estimated that millions more have yet to be identified and classified. In addition, human disciplines that explore Fungi and Bacteria are as diverse as the organisms themselves and will undoubtedly continue to grow. The relevance of microorganisms to the medical fields is more prevalent now then ever, as multi-antibiotic resistant species of infectious bacteria threaten human health. Understanding the structure and biological characteristics of infectious and non-infectious species provides key insights necessary for the creation of new antibiotic treatments and preventative strategies3-4.
Microbiology is also critical to the food industry. Numerous products, including yogurt, cheese, kombucha, bread, and alcohol are made using specific categories or species of microbes. At the same time, food scientists focus on engineering chemical compounds and preservatives that will reduce the growth of unwanted microbes on food. In all these processes, an understanding of how microbes survive, reproduce, and affect their environment is necessary.
<h4>References </h4>
<ol class="references">
	<li>Hiroshi Asakura, Holger Brueggemann, Sou-ichi Makino, and Yoshiko Sugita-Konishi, &#8220;Molecular Approaches for the Classification of Microbial Pathogens of Public Health Significance,&#8221; BioMed Research International, vol. 2014, Article ID 725801, 2 pages, 2014. https://doi.org/10.1155/2014/725801.</li>
	<li>Treseder, K. K. and J. T. Lennon (2015). 'Fungal traits that drive ecosystem dynamics on land.' Microbiol Mol Biol Rev 79(2): 243-262.</li>
	<li>Arvanitis, M. and E. Mylonakis (2015). 'Fungal-bacterial interactions and their relevance in health.' Cell Microbiol 17(10): 1442-1446.</li>
	<li>Aarestrup, F. M., et al. (2008). 'Resistance in bacteria of the food chain: epidemiology and control strategies.' Expert Rev Anti Infect Ther 6(5): 733-750.</li>
</ol>

Microbial Colony and Fungal Diversity - Concept

微生物和真菌多样性

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this ...

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Preparation of Onion Root Tips and Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">For the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water to grow roots for several days. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, clean the onion roots of any dirt or debris. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, in a 1.5 mL tube, dissolve 10 mcg of nocodazole per 1 mL of dimethyl sulfoxide solution, making 1 tube per student. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, set out all of the other tools, materials, and reagents from the list of materials onto each student&#39;s lab bench. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Prep

细胞分裂

Preparation of Onion Root Tips and Solutions
ExpandFor the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water ...

细胞结构与功能

Lab Manual Sub Articles

Bacterial Transformation

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Bacterial Transformation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">First, ensure that each student or student group has a filled ice bucket.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, set up a tube rack for each work station.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Into the tube racks, place two empty, sterile 1.5 mL tubes, and then two one mL aliquots of SOC media.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, place the plasmid tube into the ice bucket and add two tubes of competent E. coli cells, allowing them to thaw if frozen.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set out two LB agar plates per group and two LB agar ampicillin plates per group.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Ensure the students have the appropriate pipettes.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">And finally, set a water bath to 42 &#176;C and a temperature-controlled shaker to 37 &#176;C.
</div></li></ul></li></ol>

Bacterial Transformation using Plasmids - Prep

细菌转化

Bacterial Transformation
ExpandFirst, ensure that each student or student group has a filled ice bucket.
Then, set up a tube rack for each work station.
...

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Cheek Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Immediately before the experiment, wash and peel onion bulbs for the class.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Remove the entire brown outer skin and cut the onion in half with a knife. Pull apart the layers of the onion. The thin, nearly transparent film layers within the onion will be used by the students.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the onion film into a Petri dish and then place this into the refrigerator until the students arrive.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Bring out the microscopes and set them on the lab bench.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set the microscopes to the lowest magnification and plug them into an electrical outlet.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Clean the lenses with lens paper or laboratory wipes and test the knobs to make sure the lenses focus properly.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Make sure that the stage can be moved.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Prep

单元结构

Visualizing Onion and Cheek Cells
ExpandImmediately before the experiment, wash and peel onion bulbs for the class.
Remove the entire brown outer skin and ...