Name: multicolor flow cytometry-based quantification of mitochondria and lysosomes in t cells
Uploaded: 2019-01-09T14:30:00
Description: Watch this Scientific Journal Video about Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells at JoVE.com

该议定书的制定得到了台湾科技部的赠款, 台科技部提供了 nsc103-2320-010-002-my2 和 most104-2628-b-0-002-my4 至 c. l. hsu 的赠款。魏世伟是国立阳明大学微生物学与免疫学研究所优秀论文奖获得者。

这里描述的小鼠组织收获程序已获得国立阳明大学动物护理和使用机构委员会 (iacuc) 的批准。
1. 淋巴器官淋巴细胞悬浮液的制备
<ol><li>通过一种经批准的方法对小鼠进行安乐死 , 例如在透明的丙烯酸室吸入 co2, 然后进行宫颈脱位, 以确保死亡。 注: 将 co2 的流速保持在室内每分钟20% 的位移, 并且不高于 5 psi (每平方英寸磅)。鼠标需要2-3分钟才能失去知觉。?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy:+mechanisms,+pathophysiological+roles,+and+analysis.">Mitophagy: mechanisms, pathophysiological roles, and analysis.</a> Biological Chemistry. 393 (7), 547-564 (2012).</li><li>van der Windt, G. J. W., 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=CD8+memory+T+cells+have+a+bioenergetic+advantage+that+underlies+their+rapid+recall+ability.">CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (35), 14336 (2013).</li><li>Chikte, S., Panchal, N., Warnes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Lyso+dyes:+A+flow+cytometric+study+of+autophagy.">Use of Lyso dyes: A flow cytometric study of autophagy.</a> Cytometry Part A. 85 (2), 169-178 (2013).</li><li>Allan, A. 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该协议结合了有机特异染料和表面标记染色, 以量化不同 t 细胞群的线粒体或溶酶体的数量。该方法是为了克服细胞数量的限制和电子显微镜和免疫印迹分析等传统方法的同质性要求而开发的。它在分析稀有细胞群和同时检查多种细胞类型方面特别有用。
手术时间和孵化温度是该方案中最关键的因素。适当的细胞器标记需要对特定于细胞器的染料进行精确的滴定。细胞也应严...

t 细胞的激活和增殖是成功免疫反应的关键步骤。最近的进展表明, 细胞代谢与 t 细胞的发育和功能密切相关。例如, 天真的 t 细胞在很大程度上依赖氧化磷酸化 (oxphos) 来满足继发性淋巴器官循环过程中的能量需求。激活后, 天真的 t 细胞会进行剧烈的代谢重新编程, 包括诱导好氧糖酵解, 以增加 atp 的产生, 并满足细胞分化和增殖过程中的巨大代谢需求。未能通过代谢的细胞死于细胞凋亡1,2。在代谢重新编程过程中, 线粒体发挥着重要作用, 因为它们是主要负责产生 atp 的细胞器, 为细胞提供能量, 而细胞线粒体含量在代谢开关中波动在整个 t 细胞的发育和激活过程中3。然而, 多余或受损的线粒体的积累会产生多余的 ros, 损害脂质、蛋白质和 dna, 并最终导致细胞死亡
代谢改变产生的过度或受损的线粒体被一种特殊形式的自体吞噬5(称为有丝分裂) 去除。线粒体被自体染色体包裹, 然后与溶酶体融合降解。线粒体和溶酶体之间的这些密切联系引起了人们的极大兴趣。例如, 氧化应激刺激线粒体形成线粒体衍生囊泡 (mdv), 其靶向溶酶体, 以便在磷酸酶和紧张素同源体 (pten) 诱导的假定激酶 1 (pink1) 和副蛋白 (e3 泛素聚类酶) 中降解依赖方式8。研究还发现, 有丝分裂是必不可少的甜菜到白色的脂肪细胞过渡9,10。更重要的是, 溶酶体不仅是一个降解室, 也是细胞信号的调节剂。酶缺乏导致的基质积累过多, 导致溶酶体功能障碍, 破坏溶酶体膜通透性, 并影响 ca2 +稳态11。溶酶体酸脂肪酶 (lal)12或溶酶体代谢物转运蛋白敲除小鼠模型13的 t 细胞功能缺陷进一步显示溶酶体在维持 t 细胞稳态中的重要性。线粒体和溶酶体都是细胞代谢调节不可分割的部分。因此, 细胞线粒体含量的测定已成为评价 t 细胞代谢和功能状态的重要指标。
常用于量化细胞线粒体或溶酶体的内容包括免疫印迹、电子显微镜、免疫荧光 (if) 染色和线粒体 dna 拷贝数 14, 15 的 pcr 分析, 16. 虽然免疫印迹可以定量地比较不同样本的蛋白质水平, 电子或 if 显微镜可以直观地显示这些细胞器17的形态特征, 但这些检测存在一定的技术缺陷。例如, 获取足够数量的具有高放大倍率和分辨率的细胞图像, 或比较几十个样本中蛋白质的表达水平, 使这些检测被认为是低吞吐量的方法, 是非常耗时的。此外, 这些检测只能应用于同质细胞群, 如细胞系, 而不能应用于由混合群体组成的组织样本。
同样很难将这些检测方法应用于罕见的人群, 因为对这些人群来说, 从 10 6 到 108 的最低细胞数量的要求是不可能满足的。最后, 细胞通常在过程中被裂解或固定, 使其与提取进一步信息的其他方法不兼容。与传统方法相比, 基于荧光的流式细胞仪具有相对较高的吞吐量--可以同时分析和收集混合细胞群样本中所有细胞的信息。此外, 可以检测同一细胞上的10个以上的参数, 并根据所需的表型对细胞进行排序, 以便进一步检测。反应荧光探针已被用来标记溶酶体和线粒体在活细胞中, 可以通过流式细胞仪检测 18,19。这些特定于细胞的探针具有细胞渗透性, 具有物理化学特性, 使它们能够集中在特定的亚细胞位置或细胞器。方便地, 这些探头有各种荧光格式, 从而使其能够应用于多色分析。
该协议详细介绍了如何将表面标记染色与溶酶蛋白或线粒体特异性染料结合起来, 以标记活细胞中的溶酶体或线粒体。这对于从主要组织和器官中产生的样本尤其有用, 这些组织和器官通常由异质细胞群组成。研究人员可以通过其表面标记表达来识别感兴趣的细胞群, 并通过这些细胞中的有机细胞特异性染料专门测量溶酶体或线粒体含量。在这里, 我们演示流式细胞仪分析的详细过程, 评估溶酶体或线粒体质量的主要脾 t 细胞亚群。

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			<td height="21" style="height:21px;width:299px;">10 cm Graefe forceps (straight and serrated)</td>
			<td style="width:179px;">Dimeda</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">11 cm Iris scissors (straight with sharp/sharp heads)</td>
			<td>Dimeda</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">15 mL centrifuge tube</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">339650</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5 mL syringe</td>
			<td style="width:179px;">TERUMO&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">6 cm Petri dish</td>
			<td style="width:179px;">&#945;-plus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">70 &#181;m nylon cell strainer</td>
			<td style="width:179px;">SPL Lifesciences</td>
			<td style="width:119px;">93070</td>
			<td style="width:167px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;">Ammonium chloride (NH4Cl) &#160;&#160;</td>
			<td>Sigma</td>
			<td style="width:119px;">A9434</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD25</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">102007</td>
			<td style="width:167px;">Clone:PC61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD4</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">100453</td>
			<td style="width:167px;">Clone:GK1.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD44</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">103029</td>
			<td style="width:167px;">Clone:IM7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD62L</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">104407</td>
			<td style="width:167px;">Clone:MEL-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse CD8</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">100713</td>
			<td style="width:167px;">Clone:53-6.7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-mouse TCR&#946;</td>
			<td style="width:179px;">Biolegend</td>
			<td style="width:119px;">109233</td>
			<td style="width:167px;">Clone:H57-579</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">BD LSRFortessa</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:179px;">Bio basic</td>
			<td style="width:119px;">6381-92-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FALCON 5ml Polystyrene Round-Bottom Tube (FACS tube)</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;">352052</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FcRgamma II (CD32) Hybridoma (2.4G2)</td>
			<td style="width:179px;">ATCC</td>
			<td style="width:119px;">HB-197&#12288;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:179px;">Hyclone</td>
			<td>ATD161145</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Flowjo, LLC</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydroxyethyl piperazineethanesulfonic acid (HEPES)</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td style="width:179px;">Gibco</td>
			<td>A2916801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">LysoTracker Green DND26</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L7526</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">MEM Non-essential amino acids (100X)</td>
			<td style="width:179px;">Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">MitoTracker Green FM</td>
			<td style="width:179px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">M7514</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:179px;">Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium bicarbonate (KHCO3)</td>
			<td style="width:179px;">J.T.baker</td>
			<td style="width:119px;">298-14-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Propidium iodide solution</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">25535-16-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">RPMI 1640 medium (powder)</td>
			<td style="width:179px;">Gibco</td>
			<td>31800089</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium pyruvate (100 mM)</td>
			<td style="width:179px;">Gibco</td>
			<td>11360070</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

multicolor flow cytometry-based quantification of mitochondria and lysosomes in t cells

t 细胞利用不同的代谢程序来满足它们在分化和增殖过程中的功能需求。线粒体是负责提供细胞能量的关键细胞成分;然而, 过量的线粒体也会产生活性氧 (ros), 从而导致细胞死亡。因此, 线粒体的数量必须不断调整, 以适应细胞的需要。这种动态调节在一定程度上是通过溶酶体的功能实现的, 溶酶体去除了表面损伤的细胞器和大分子。因此, 细胞线粒体和溶酶体含量是评估细胞代谢调节的关键指标。随着细胞器探针的发展, 有良好特征的溶酶体或线粒体特异性染料已成为可用的各种格式, 以标记细胞溶酶体和线粒体。多色流式细胞术是一种常用的工具来描述细胞表型, 并具有与其他检测集成的能力。在这里, 我们提出了一个详细的协议, 如何结合有机特定染料与表面标记染色, 以测量流式细胞仪上不同 t 细胞群中的溶酶体和线粒体的数量。

脾脏和胸腺主要 t 细胞亚群的鉴定
总之, 脾脏和胸腺的单细胞悬浮液由红细胞裂解, 用 2.4 g2 上清液孵育, 然后用荧光共轭抗体进行有机染料和表面标记染色 (表 1)。胸腺细胞的发育过程可以用抗体来描述共同受体 cd8 和 cd4。最不成熟的胸腺细胞不表达 cd4 或 cd8, 被称为双阴性 (dn)。然后, 它们上调 cd4 和 ...

Watch this Scientific Journal Video about Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells at JoVE.com

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

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。

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

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。

Research

JoVE Journal

Immunology and Infection

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

多色流流式细胞仪分析细胞在恒河猴的免疫应答

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

科研

免疫与感染

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

流式细胞仪用来评估染色质的T细胞国家

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

小鼠肾-居民CD8+T 细胞的分离，用于流细胞测量分析

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...