Immunology and Infection
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基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量
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Summary January 9th, 2019
Please note that all translations are automatically generated.
Click here for the English version.
本文说明了一种对活细胞中线粒体或溶酶体进行量化的有力方法。将溶酶体或线粒体特异性染料与针对表面标记物的荧光共轭抗体结合使用, 可以通过使用多色流式细胞术。
Transcript
该方法可以帮助我们回答免疫代谢领域的关键问题。这种技术的优点是，它允许在不同细胞类型的复杂混合物中对单个活细胞中的线粒体或解索体进行定量。这种研究T和B细胞的方法也可以应用于许多其他细胞类型，如巨噬细胞，树突状细胞，或从组织样本中收获的任何初级细胞。
这个程序将由我实验室的研究生魏钦文演示。要标记细胞器进行定量，首先，将十倍添加到第六个小鼠T细胞中，每个标记的一个圆底传真管中。通过离心对细胞进行造粒。
预热至37摄氏度无血清培养基，稀释细胞器特异性探针库存溶液，最终在培养基中工作浓度。并每管将100微升探针染料的细胞重新悬浮。接下来，在细胞培养培养箱中孵育细胞，以适当的染色期，每管用一毫升的冰冷传真缓冲液停止孵化结束时的反应。
然后，用另一种离心收集细胞，并在冰上预滴答的24G2杂交上先液的100微升中重新悬浮颗粒。十分钟后，每管添加一毫升传真缓冲液。通过离心收集细胞，并标记细胞与50微升的预滴定荧光结合抗体鸡尾酒的兴趣。
根据表格添加适当的浓度。和传真缓冲20分钟的冰上，防止光。在孵育结束时，每管添加一毫升传真缓冲液。
通过离心收集细胞。并在300微升传真缓冲液中重新悬浮细胞，并辅以每毫升碘化铀1微克。添加核和染色体计数器后，立即打开流式细胞仪分析计算机、流式细胞仪、传真采集软件以及任何其他附件设备，具体取决于机器平台。
通过系统运行 PBS 约一分钟，以确保流线充满 PBS 和 sheathe 缓冲区。打开新实验，根据制造商的说明设置圆度计参数。通过70微米细胞滤株过滤细胞。
并在采集每个样品之前出现涡流，以避免团团和双层形成。打开"自动补偿"设置，并为每个荧光参数创建点图。设置所有电压后，调整补偿并应用补偿对样品。
创建向前散射与侧散射图并优化电压，以便感兴趣的单元出现在图中。创建向前散射与向前散射高度图，并门控单个单元。创建一个向前散射区域与侧散射区图，并门对淋巴细胞。
创建一个向前散射区域与碘化铀图，并门对活细胞。设置所有电压后，调整补偿并应用补偿对样品。然后加载第一个样本管，创建适当的细胞表面标记图，并收集至少 1，000 个感兴趣的总体事件。
运行所有示例后，导出流数据以进行后续分析。将实验保存为模板，以保留未来类似实验的细胞计设置和参数。最终位点线粒体含量为双负一T细胞中最低的，双负一T细胞的峰数为双负二、三，双负四阶段略有下降。
双阳性胸腺细胞显示线粒体数量高于CD4和CD8单阳性胸腺细胞，表明线粒体含量在发育过程中波动。CD4 和 CD8 单阳性胸腺细胞表现出比单体 CD4 或 CD8 T 细胞更高的线粒体平均荧光强度，这表明在富氧血液环境中再循环的天真 T 细胞的线粒体质量甚至比胸腺细胞低。有趣的是，这种线粒体含量的减少在CD8中比CD4 T细胞系更加突出，T细胞激活进一步减少了这些细胞器的存在。
在所有胸腺细胞群中观察到相对较低但可检测的利索马血素含量，在双阴性一个胸腺细胞中存在更突出。在外围，在记忆和效应CD8阳性T细胞中检测到相对大量的利索索姆。将细胞器特异性染料和表面标记物与流式细胞学相结合，是描述复杂混合物中细胞代谢状态的有力方法。
额外的细胞器特异性染料可用于检测内质神经，自噬性气囊，或其他细胞内感兴趣的隔间。
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