This work is supported by the Joseph Alexander Foundation, the ASBMB Undergraduate Research Award, 2013-2014, and a Science Award Grant, Marymount Manhattan College, 2012-2013.

注：操守II级生物安全柜的所有工作与细胞和细胞培养试剂（BSC）9。 MMT细胞被列为生物安全I级，因为它们对低到中等风险的生物。应用适当的清洁和消毒程序到BSC用途（ 例如 ，紫外光，70％的乙醇擦拭）之间。 1.成长MMT细胞<ol><li>生长的细胞在含有10ml补充有10％胎牛血清（FBS）的营养丰富的媒体，它由鹰最低基本培养基 （EMEM），2mM谷氨酰胺和1％抗真菌/抗菌10厘米组织培养皿（10个单位的青霉素，100微克链霉素，0.25微克两性霉素B）。培养的细胞在湿润的5％CO 2的室中，在37℃下。板的细胞在3.6×10 6个细胞/ cm 2的密度（参见步骤2细胞计数）。 </li><li>更换一半的细胞培养基用新鲜培养基每48小时UNLESS的另外指出。检查细胞存活力和形态由检查用倒置相差光学显微镜。 <ol><li>注下在100的尺寸，结构，形状，组织和预计数量表征细胞 - 放大200倍。在此密度，细胞应该出现在一个平面上，而不是结块的顶上彼此接近。每个小区由一个厚，球形芯薄，长，分支状延伸，从它那来的点扩展（参见图1）。 </li></ol></li><li>细分细胞，当他们到达7.2×10 6个细胞/ cm 2的细胞密度。细胞表现出1.8×10 6个细胞/ cm 2的每24小时的倍增速率。指定细胞通道X（过氧化物酶）来表示的时候，他们已经被拆分的数量。 <ol><li>在第3代（ 即 ，经过三次传代，P3）收获，计数，和板的细胞到一个24孔组织培养板上以3.6×10 6的密度</s达&gt;细胞/ cm 2。 </li></ol></li><li>每天记录的数字显微照片查看细胞。注意，描述细胞形态（ 即 ，大小，形状，光反射特性）。通过定期计数细胞并与经过的时间，以细胞数测定细胞倍增时间。 </li></ol> 2.计数细胞MMT 注意：计数细胞，以确定是否细胞需要传代培养，以设置一个实验，或确定细胞活力。还有这里介绍两种方法。 <ol><li>使用血细胞计数器的，一个标准的技术来计数细胞。 <ol><li>获得光明行血细胞计数，台盼蓝染色，复合光显微镜，无菌试管，移液器和手动细胞计数0.2％的溶液。 </li><li>根据BSC，用10毫升吸管从10cm的组织培养皿中取出的细胞。如果细胞生长在24孔板用1毫升吸管或1000微升微量去除媒体。 </li><li&gt;绘制细胞介质插入吸管，将针对该培养皿的底部的吸移管的前端排出的媒体滑动的同时沿板枪头。重复4 - 5倍，以帮助去除细胞，分离块和导致更准确的细胞计数。 </li><li>放置所得到的细胞悬浮液在一个15毫升无菌试管（或1毫升微离心机或其他小体积管）。否则，保留的细胞在原始组织培养板/孔。 </li><li>下在BSC，结合10微升细胞悬浮液与10微升的台盼蓝溶液的：在一非无菌微量离心或其他小体积管（1比1的比例）。 注意：台盼蓝是不是由健康的活细胞吸收了活力染色。当细胞受损或死亡，台盼蓝可以进入细胞使死细胞计数（又名染色排除法 ）。因此，在显微镜下，死细胞出现暗紫色的，而活细胞是明亮，颜色浅。 </li><li>孵化在RT 5分钟。 </li><li>广场上的血球盖玻片。申请10微升台盼蓝细胞悬浮混合物的进入凹槽。查看血球在100 - 200倍。一个四角落网格是表观（见图2）。 </li><li>计数存活的细胞（未染色）对细胞总数在每个网格区域的数量。平均四个网格和由1×10 4相乘，以获得细胞/ ml。 <ol><li>推断每皿的细胞（或细胞/ cm 2）的总数量。使用此号码或者确定所述细胞悬浮液的正确的卷使用的种子的新的组织培养板上以3.6×10 6个细胞/ cm 2的期望的密度或确定在平板上或者在井的活细胞的总数。 注：有关使用血球的更多指导，请参阅10。 </li></ol></li></ol></li><li>使用软件和数字照相机计数活细胞。 <ol><li>获得二gital摄像头，与之配套的软件，台盼蓝染色，复合光显微镜，无菌试管，移液管和一台电脑一个0.4％的溶液。 注：Moticam 2000数码相机附带的软件麦克奥迪这里是就业。任何类似的相机和软件就足够了。确保数字相机软件已经校准提前（根据制造商的协议）。 </li><li>根据BSC，用1毫升吸管或1000微升微量从MMT细胞的24孔组织培养皿成长取出介质。轻轻施加少量（约500微升）的未补充（即 EMEM无任何补充剂）的新鲜培养基到板然后除去它洗贴壁细胞。直接向好：应用10微升0.2％台盼蓝（1与联合国补充EMEM稀释制成1）。 </li><li>孵育板在RT 5分钟。 </li><li>查看在显微镜下100盘 - 200X倍率W¯¯第i个数字照相机附。板可以用未补充的媒体，如果用2％台盼蓝染色太暗。 </li><li>在电脑上一打开软件ND验证软件校准通过设置选项卡中使用的目标。视野的图像应该会出现。 </li><li>从测量选项卡的网格 。正方形的格子​​约为0.005Hz厘米宽将出现。选择网格信息以确认每平方米的面积。 </li><li>选择网格的一个特定区域，计数细胞（ 即 ，5平方×9正方形）。从测量选项卡中选择矩形 。点击广场一角，并拖动光标涵盖选择的平方。 注意：绿色的灯罩将涵盖的选择，并且白框将显示在提供的宽度，高度，区域的拐角处，和所选择的网格的部分的周长的平方（ 见图3）。 </li><li>算VIAB数所确定的区域内的文件的细胞。通过在显微镜下移动板做相同的同井或板2的其它位置。 </li><li>可以肯定的测量面积是相同的，并且放大倍率也没有改变。计算面积内的活细胞的平均数量。使用井的区域（24孔板，一个孔具有2 cm 2的面积，对于100mm培养皿面积78.6平方厘米 ），推断活细胞的数量从划定在网格中的区域内的孔（细胞/ cm 2）的细胞的总数。 </li></ol></li></ol> 3.治疗MMT细胞具有抗增殖剂<ol><li>制备所选择的抗增殖治疗剂（他莫昔芬，姜黄素和二甲双胍）和任选的药物，阿司匹林在BSC下的解决方案。 <ol><li>溶解姜黄素和他莫昔芬在100％乙醇，以产生一个库存浓度为27毫摩尔。溶解二甲双胍和阿司匹林unsupplement编EMEM以产生原料浓度500mM的和15mM的分别。 </li></ol></li><li>建立剂量反应。 <ol><li>治疗MMT细胞与三个抗增殖治疗剂（他莫昔芬，姜黄素和二甲双胍）和任选的药物（阿司匹林）在不同浓度的96小时，以产生剂量反应曲线。最初管理所有药物在一个范围的基础上发表的报告1,11-16浓度，然后在浓度比出版更大或更小。 注意：剂量反应确定需要产生所期望的结果的药物的最低浓度。这里所期望的结果作为相对于对照中的细胞增殖的减少。 <ol><li>对于他莫昔芬和姜黄素，使用0.054毫米（1微升），0.108毫米（2微升），0.162毫米（3微升）和0.216毫米（4微升）的浓度（以及相应的卷）。 </li><li>对于二甲双胍，使用2mM的浓度（和相应的体积）（2微升），4mM的（4微升），6毫（6微升），8毫（8微升）和10mM（10微升）。 </li><li>为阿司匹林，使用0.030毫摩尔（1微升），0.060毫（2微升），0.099毫（3.3微升），0.150毫（5微升）和0.216毫米（6.7微升）浓度（和相应的卷）。 </li></ol></li><li>分割从10cm皿MMT细胞到24孔板中以3.6×10 6个细胞/ cm 2的浓度。确定由这两个细胞计数的方法（步骤2）初始细胞浓度。呼叫细胞这种新的24孔板“ 日拆分”。 </li><li> 24小时细胞铺板后，治疗的MMT细胞与每个抗增殖治疗剂，他莫昔芬，姜黄素，二甲双胍和阿司匹林，在步骤3.2.1中描述的浓度。称此为“0天 ”。 <ol><li>由于每口井可以容纳最多的新井进行处理，以避免CR每次500微升介质，使用微量用无菌微管提示和技巧的新OSS污染。使用两个井作为对照：在没有任何药物治疗（阴性对照）和细胞中存在的100％的乙醇（溶剂对照）生长的细胞生长。 </li></ol></li><li>生长细胞并在所描述的浓度重新给予药物时，供给细胞每隔一天（参见步骤1.2）96小时（直到第4天）在天1 - 4治疗，观察细胞的显微镜下，并使用计数在步骤2.2中所述的方法（参见图4）。 </li><li>重复实验至少三次。 </li><li>通过作图在实验的长度细胞活力和药物剂量之间的关系确定每种药物的最佳浓度（参见图5）。 </li></ol></li><li>建立一个时间过程。 <ol><li>治疗MMT细胞与三个抗增殖治疗剂（他莫昔芬，姜黄素和二甲双胍）在一个固定的浓度对不同时间段。使用最佳浓度IDENTIF通过剂量反应实验IED（见步骤3.2）。 <ol><li>使用下列浓度：0.216毫三苯氧胺，0.216毫姜黄素和10mM二甲双胍。 注意：时间过程确定的必要时间量的药物，以产生其最佳期望的结果。这里，所期望的结果作为相对于对照中的细胞增殖的减少。 </li></ol></li><li>分割从10cm皿MMT细胞到24孔板中以3.6×10 6个细胞/ cm 2的浓度。确定由这两个细胞计数的方法（步骤2）初始细胞浓度。呼叫细胞这种新的24孔板“ 日拆分”。 </li><li> 24小时细胞铺板后，治疗的MMT细胞与每个抗增殖治疗剂，他莫昔芬，姜黄素，二甲双胍和阿司匹林，在步骤3.2中确定的最佳浓度。称此为“0天”。 <ol><li>由于每口井可以容纳最多500微升的媒体，美国Ë微量用无菌微量的技巧和新井进行处理，以避免交叉污染，每次一个新的提示。 </li><li>使用两个井作为对照：在没有任何药物治疗（阴性对照）和细胞中存在的100％的乙醇（溶剂对照）生长的细胞生长。 </li></ol></li></ol></li><li>生长细胞并在所选择的浓度重新给予药物时，供给细胞每隔一天（参见步骤1.2）96小时（直到第4天）。关于天1 - 4治疗，观察细胞的显微镜下，使用该方法在步骤2.2中（ 见图4）计数。 </li><li>重复实验至少三次。 </li><li>通过作图在所选浓度细胞活力和药物治疗的长度（时间）之间的关系确定MMT细胞每种药物的最佳曝光时间（参见图6）。 </li></ol> 4.实验室模块注：以下是5天为实验室模块。 图7实验室时间表是什么样的时间表将内5天带来的流程图。对于此活动到五天内完成任一时间过程或剂量响应曲线被产生。没有足够的时间来生成曲线。下方的剂量反应实验说明。时程实验可容易地互换<ol><li>分裂类分组：有一组建立剂量响应和其它的时间过程中选择在建议的剂量反应浓度的中间浓度。 <ol><li>保持足够的文化和药物库存在适当的浓度。除非另有说明，执行BSC下的所有工作。 </li></ol></li><li>第1天<ol><li>直接学生作出媒体和生长的细胞（如在步骤1）。 </li><li>由于学生获得MMT细胞的股票在10厘米板，引导学生准备细胞培养基，并给一个教程中使用hemocytome的之三，数码显微镜和数码相机显微镜软件（如步骤2）。 </li><li>校准软件（ 例如，4X，10X，20X）现在使用的制造商的协议在显微镜中使用的目标。 </li></ol></li><li>第2天<ol><li>有学生确认总细胞数目和细胞存活力（如在步骤2）。 </li><li>有学生计数细胞上同时使用显微镜软件和血球的方法，确认了类似的结果，得到的100mm培养皿。 </li><li>直接学生获得从100mm培养皿于24孔板，以所希望的起始细胞铺板密度24孔板和分裂的细胞（如在步骤1）。这被认为是节分割 。将24孔板中的细胞培养孵化24小时。 </li></ol></li><li> 3天<ol><li>生长MMT细胞上24孔板24小时。让学生观察细胞的显微镜下，用显微镜软件（如步骤2）计算。 </li><li>给学生/ TE是癌症治疗药物的股票样本。让他们准备和稀释原始股票的解决方案（如步骤3）。各种浓度的药物添加到其细胞以建立剂量响应曲线。这被称为第0天 。 </li><li>孵育细胞（步骤1.2）与药物最多48小时。 </li></ol></li><li> 4天<ol><li>让学生在24小时后观察治疗的细胞。这是第1天 。 </li><li>每个计数采用公显微镜软件。每个记录的照片以及使计数可以在实验室外进行（如在步骤2）。 </li><li>孵育细胞（步骤1.2）与药物另外24小时。 注：教师提供额外的指南和评论数据分析和图形演示。学生们学习如何创建人物，情节，以及统计分析数据收集的第二天。 </li></ol></li><li>第5天<ol><li>已经48小时后，2日的学生处理的细胞。 </li><li>每个计用好显微镜软件。每个记录的照片以及使计数可以在实验室外进行（如在步骤2）。 </li><li>收获和收集细胞用于通过传统的血细胞计数方法验证有效地利用显微镜软件方法学生能力的手段计数（如步骤2）。 注：收集数据，并得出结论或修改假设，相应。 </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+and+cancer:+an+'old-age'+disease+with+an+'age-old'+solution.">Curcumin and cancer: an 'old-age' disease with an 'age-old' solution.</a> Cancer Lett. 267 (1), 133-164 (2008).</li><li>Ararat, E., Sahin, I., Altundag, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+behind+the+aspirin+use+and+decreased+breast+cancer+incidence.">Mechanisms behind the aspirin use and decreased breast cancer incidence.</a> J BUON. 16 (1), 180 (2011).</li><li>Ararat, E., Sahin, I., Altundag, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspirin+intake+may+prevent+metastasis+in+patients+with+triple-negative+breast+cancer.">Aspirin intake may prevent metastasis in patients with triple-negative breast cancer.</a> Med Oncol. 28 (4), 1308-1310 (2011).</li><li>Blandino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metformin+elicits+anticancer+effects+through+the+sequential+modulation+of+DICER+and+c-MYC.">Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC.</a> Nat Commun. 3, 865 (2012).</li><li>Kunnumakkara, A. B., Anand, P., Aggarwal, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+inhibits+proliferation,+invasion,+angiogenesis+and+metastasis+of+different+cancers+through+interaction+with+multiple+cell+signaling+proteins.">Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins.</a> Cancer Lett. 269 (2), 199-225 (2008).</li><li>Burstein, D. E., Blumberg, P. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor-induced+neuronal+differentiation+of+PC12+pheochromocytoma+cells:+lack+of+inhibition+by+a+tumor+promoter.">Nerve growth factor-induced neuronal differentiation of PC12 pheochromocytoma cells: lack of inhibition by a tumor promoter.</a> Brain Res. 247 (1), 115-119 (1982).</li><li>Nazarali, S. A., Narod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tamoxifen+for+women+at+high+risk+of+breast+cancer.">Tamoxifen for women at high risk of breast cancer.</a> Breast Cancer (Dove Med Press). 6, 29-36 (2014).</li><li>Cui, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-talk+between+HER2+and+MED1+regulates+tamoxifen+resistance+of+human+breast+cancer+cells).">Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer cells).</a> Cancer Res. 72 (21), 5625-5634 (2012).</li><li>Komm, B. S., Mirkin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+current+and+emerging+SERMs.">An overview of current and emerging SERMs.</a> J Steroid Biochem Mol Biol. 143C, 207-222 (2014).</li><li>Kaplan, R. M., Satterfield, J. M., Kington, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+better+physician--the+case+for+the+new+MCAT.">Building a better physician--the case for the new MCAT.</a> N Engl J Med. 366 (14), 1265-1268 (2012).</li></ol>

作者没有收到来自麦克奥迪财务或类似的支持。

实验室模块提出，旨在通过动物细胞培养的先进技术传授细胞生物学的各种话题。模块通过分析若干抗增殖化学品对细胞模拟人类乳腺癌的复制的效果达到这一点。主要检测依赖于细胞计数的基本技术，并引入了一种新的方法来计算使用显微镜的软件单元。包括该模块的活动可以与仪器和设备，在大多数生物学程序可用来进行。该模块可在5天的日程与用品是廉价且容易地得到实现。虽然数码显微...

往往在普通本科生物学课程，细胞周期调控和癌症的主题上眼，但没有详细探讨，因为内容在这些课程的广度留下一点时间深入。此外，本科学生的生物通常不会暴露在与动物细胞培养相关的先进技术。为了帮助学生养成对这些概念有了更深的了解，同时应用和分析所学，实验室活动是作为研究的沃尔特·里德陆军研究所 （WRAIR）扩展实验活动1的修改。实验室模块使用逐步的，实验策略，其包括生长和表征的癌细胞模型，开发和执行细胞计数的方法，确立最佳时间进程和剂量与抗增殖剂处理的细胞，并确定异常细胞信号传导途径。实验还允许开-ended询问。 大多数需要这种活动的技术可以实现在一个典型的生物学教学实验室。活动开始与学生表征的小鼠乳腺肿瘤（MMT）细胞系的形态和生长速率，模型为人类乳腺癌2。乳腺癌的选择，因为它的人口患病率，其熟悉大学年龄的学生，和现有的广泛的数据作为模型癌症。在MMT细胞系特别选择，因为它是容易获得的，良好表征的，具有短倍增时间，容易成长。此外，MMT细胞是雌激素依赖这与大多数女性乳癌一致。学生然后通过与化疗药物，其作用机理是很好的药物和的处理长度的确立。浓度改变允许学生处理细胞识别所述MMT细胞异常细胞信号传导途径评价这些变量对细胞分裂的速率的影响。重点分析了本次活动是细胞活力，它只是需要细胞计数，使用以下两种方法之一的决心。每种方法依赖于强大的显微镜技术。学生确定细胞存活率通过使用标准的，血球的方法和一种新的显微照相方法和建议。根据他们的研究结果，他们可以提出并测试修改的活性。然后学生表示他们的数据和解释结果，以完善他们的假设，并制定新的实验策略。 该实验室的活动适合于大一大二或水平的学生主修生物科学。它被冷凝成可在第一年完成，一般生物学或第二年，蜂窝/分子生物学过程的一周实验室模块。需要正确完成活动的技能包括基本的算术和代数，熟悉的C数组矿石实验室技能（ 例如 ，移液，溶液制作，无菌技术），数据分析，基本光镜和时间管理，以及细胞培养和电子表格软件的讲师知识。所需试剂包括的动物细胞系模型对癌症（ 例如 ，小鼠乳腺肿瘤细胞，MMT 2），化疗剂（ 例如 ，他莫昔芬，姜黄素，二甲双胍，和阿司匹林），台盼蓝和细胞培养基（ 例如，Eagle氏最低必需培养基 ; EMEM）用适当的补充剂（ 例如 ，供体马和牛胎儿血清）。所需仪器包括一个倒置光学显微镜用数码相机连接，计算机100毫米和24孔组织培养板，CO 2培养箱（或等效物），生物安全柜（BSC; II级），血球和数字显微镜软件。 也有特定的实验室活动依赖于动物细胞培养TEAC很好的例子^ h本科生约在细胞生物学3概念。然而，许多需要物资或技术是不容易（ 例如 ，放射性同位素，活的动物组织，先进的影像设备1,4,5），描述是相当先进（ 例如 ，适合400级课程6）协议，或需要多周或学期长的项目6,7。这里描述的实验活动很简单，可与常见的实验室设备一个星期进行。 综上所述，本实验模块有效地引入或增强细胞周期，细胞信号通路与肿瘤的概念，而教学的基本和高级技能实验室，实验数据分析，动物细胞培养的方法和科学的过程。该实验室模块简单，经济方便和开放式查询提供了灵活性和机会。该活动鼓励学生的创造力通过提供模板实验策略，作为一个指导，但不是一个配方。最重要的是，该活动满足了花开分类8所有学习领域，因为它需要记忆，理解，应用，分析，评估和创建通过参与学生在过程中，拉他们走出课本，走进科研的世界。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>ESCO Labculture Reliant</td>
			<td></td>
			<td>Class II Type A2 Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Waterjactor CO2 Incubator</td>
			<td>CEDCO</td>
			<td>Model 1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-line Hemocytometer</td>
			<td>American Optical</td>
			<td></td>
			<td>with two separate grids</td>
		</tr>
		<tr>
			<td>Motic Images Plus</td>
			<td></td>
			<td></td>
			<td>Mac OSX Verison 2.0 or higher</td>
		</tr>
		<tr>
			<td>Gilson Pipetman</td>
			<td>Rainin instrument co. inc</td>
			<td></td>
			<td>P-20D, P-200D, P-1000D</td>
		</tr>
		<tr>
			<td>CK30/CK40 Culture Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>4 objective inverted light microscope with camera</td>
		</tr>
		<tr>
			<td>200 uL Pipet tips</td>
			<td>MidSci</td>
			<td>40200C</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 uL Pipet tips</td>
			<td>MidSci</td>
			<td>AVR4</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Seriological Pipets</td>
			<td>TPP</td>
			<td>TP94010</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>CoStar- Tissue Culture Cluster</td>
			<td align="right">3524</td>
			<td>24 wells, 16 mm well diameter, Radiation sterilized</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>100 mL, cell culture tested non-haz</td>
		</tr>
		<tr>
			<td>Bright-line Hemacytometer replacement coverslip, non-haz</td>
			<td>Sigma</td>
			<td>Z375357</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Mammary Tumor(MMT) cells</td>
			<td>ATCC</td>
			<td>CCL-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle Minimum Essentail Medium (EMEM)</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F0926</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Meformin Hydrochloride</td>
			<td>Sigma</td>
			<td>PHR1084</td>
			<td>500 mg</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>white or white-yellow powder</td>
		</tr>
		<tr>
			<td>Curmumin</td>
			<td>Sigma</td>
			<td>C1386</td>
			<td>yellow-orange powder</td>
		</tr>
		<tr>
			<td>Aspirin</td>
			<td>Sigma</td>
			<td>A2093</td>
			<td>meets USP testing specifications</td>
		</tr>
	</tbody>
</table>

using mouse mammary tumor cells to teach core biology concepts: a simple lab module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

成长MMT细胞和比较计数方法 。 小鼠乳腺肿瘤细胞成功地生长和特征（ 图1），并使用麦克奥迪软件，用于显微镜的数码相机相关的软件程序开发了一种新的细胞计数法。这种新的细胞计数法进行比较采用血细胞计数器（ 图2）传统的计数方法，被证明是同样准确确定细胞数目（ 表1）。为了控制由于不同的生长条件或细胞类型上的...

Watch this Scientific Journal Video about Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module at JoVE.com

Using Mouse Mammary Tumor Cells to Teach Biology Concepts | Biology | Video

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

用小鼠乳腺肿瘤细胞教生物学的核心概念：一个简单的实验模块

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

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12.8K 次观看.  Marymount Manhattan College. A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

视频: Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

科研

生物学

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

乳腺上皮移植手术

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

导管内注射局部给药的小鼠乳腺

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

实验教学中乳糖酶活性的测定

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...