我们要感谢 Dr. Jue Liu 的热心帮助。本研究得到了国家重点研发计划（编号：2022YFD1800300）、国家自然科学基金（编号：32130105）和现代涉农产业技术研究体系专项资金（编号.CARS-40）、中国。

材料 表中列出了研究中使用的试剂和设备的详细信息。
1. 样品池的制备
<ol><li>在 38 °C 的 5% CO2 培养箱中，用补充有 10% 胎牛血清 （FBS） 的 Dulbecco 改良 Eagle 培养基 （DMEM） 在六孔板（每孔 5 x 105 个细胞）中培养 DF-1（永生鸡胚胎成纤维细胞）细胞。</li>
	<li>当细胞达到 80% 汇合时，弃去含血清的细胞培养基，用磷酸盐缓冲盐水 （PBS） 洗涤细胞 3 次，然后向每个孔中加入 2 mL 无血清细胞培养基。</li>
	<li>向每个孔中加入 IBDV 病毒溶液（含有计算的 MOI），并通过添加等于病毒溶液体积的 DMEM 来设置模拟感染的对照。</li>
	<li>病毒吸附 1 小时后，弃去培养基，用 PBS 洗涤细胞 3 次，向每个孔中加入 2 mL 补充有 2% FBS 的 DMEM，并继续细胞培养 24 小时。</li>
	<li>IBDV 感染后 24 小时，进行胰蛋白酶处理（每孔 500 μL）1 分钟，然后通过补充 10% FBS（每孔 2 mL）的 DMEM 中和。之后，必须收获细胞以制备用于流式细胞术的单细胞悬液。</li>
	<li>将细胞在 2-8 °C 下以 400 x g 离心 5 分钟。 使用移液管弃去上清液。</li>
	<li>将细胞沉淀重悬于 PBS 中，并轻轻涡旋试管 3 秒。</li>
	<li>如步骤1.6所述离心细胞（在2-8°C下400 x g 5分钟）。</li>
	<li>如步骤 1.7 和步骤 1.8 中所述重复细胞洗涤。</li>
	<li>在显微镜下使用血细胞计数器进行细胞计数。然后，将细胞重悬于适当体积的流式细胞术染色缓冲液中（使最终细胞浓度为 1 x 107 个细胞/mL）。轻轻涡旋悬浮液。</li>
</ol>2. 用于流式细胞术的细胞染色
<ol><li>将步骤 1.10 中的 100 μL 单细胞悬液添加到 5 mL 圆底聚苯乙烯管（12 mm × 75 mm）中。准备用于抗 chGSDME-NT/CT 抗体染色的模拟和 IBDV 感染的细胞，并使用正常小鼠 IgG 作为同种型对照。同时，用 IBDV 感染的细胞准备空白对照和单染色管。</li>
	<li>分别向 IBDV 感染管和模拟对照管中加入 1 μg Alexa Fluor 647 标记的抗 chGSDME-NT MCAB、抗 chGSDME-CT MCAB 或正常小鼠 IgG（作为同种型对照）。随后，轻轻涡旋试管 3 秒。</li>
	<li>将染色的细胞在 2-8 °C 或冰上避光孵育 30 分钟。每 10 分钟轻轻涡旋试管。</li>
	<li>用 1 mL 流式细胞术染色缓冲液在 2-8 °C 下以 400 x g 离心 5 分钟洗涤细胞。 使用移液管弃去上清液。</li>
	<li>如步骤 2.4 中所述重复细胞洗涤。</li>
	<li>将沉淀重悬于 100 μL 流式细胞术染色缓冲液中。</li>
	<li>在室温下用 10 μg/mL 的 PI 对 IBDV 感染组和模拟感染组的细胞以及 PI 单染色管染色 10 分钟，然后加入 400 μL 流式细胞术染色缓冲液用于流式细胞术测定。</li>
</ol>3. 进行流式细胞术检测
<ol><li>打开流式细胞仪以初始化仪器并启动软件。</li>
	<li>首先运行空白对照管，然后运行单根染色管，以调整流式细胞仪的电压和补偿参数。利用在 635 nm 处发射的 FL3 和 FL4 荧光通道，分别检测 PI 和 Alexa Fluor 647 荧光基团。</li>
	<li>配置完所有参数后，按顺序运行所有样本。</li>
</ol>4. 流式细胞术数据分析
<ol><li>分析 FL4 通道直方图中每个样品的 Alexa Fluor 647 染色。</li>
	<li>为了评估每个样品中双阳性细胞的比例，请使用 FL3/FL4 的四象限点图。</li>
	<li>根据同种型对照 IgG/PI 双染样品设置阳性阈值，确保双阳性细胞的比例保持在 1% 以下。这种方法有助于测量所有样品中双阳性细胞的比例。</li>
</ol>

定量传染性法氏囊病病毒感染的 DF-1 细胞中的焦亡

<ol>
	<li>He, W. T., 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=Gasdermin+D+is+an+executor+of+pyroptosis+and+is+required+for+interleukin-1&#946;+secretion.">Gasdermin D is an executor of pyroptosis and is required for interleukin-1&#946; secretion.</a> Cell Res. 25 (12), 1285-1298 (2015).</li><li>Kayagaki, N., 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=Caspase-11+cleaves+Gasdermin+D+for+non-canonical+inflammasome+signalling.">Caspase-11 cleaves Gasdermin D for non-canonical inflammasome signalling.</a> Nature. 526 (7575), 666-671 (2015).</li><li>Shi, 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=Cleavage+of+GSDMD+by+inflammatory+caspases+determines+pyroptotic+cell+death.">Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.</a> Nature. 526 (7575), 660-665 (2015).</li><li>Angosto-Bazarra, D., 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=Evolutionary+analyses+of+the+Dasdermin+family+suggest+conserved+roles+in+infection+response+despite+loss+of+pore-forming+functionality.">Evolutionary analyses of the Dasdermin family suggest conserved roles in infection response despite loss of pore-forming functionality.</a> BMC Biol. 20 (1), 9 (2022).</li><li>De Schutter, E., 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=Punching+holes+in+cellular+membranes:+Biology+and+evolution+of+gasdermins.">Punching holes in cellular membranes: Biology and evolution of gasdermins.</a> Trends Cell Biol. 31 (6), 500-513 (2021).</li><li>Kovacs, S. B., Miao, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gasdermins:+Effectors+of+pyroptosis.">Gasdermins: Effectors of pyroptosis.</a> Trends Cell Biol. 27 (9), 673-684 (2017).</li><li>Jiang, S., Gu, H., Zhao, Y., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teleost+gasdermin+E+is+cleaved+by+caspase+1,+3,+and+7+and+induces+pyroptosis.">Teleost gasdermin E is cleaved by caspase 1, 3, and 7 and induces pyroptosis.</a> J Immunol. 203 (5), 1369-1382 (2019).</li><li>Li, H., 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=Duck+gasdermin+E+is+a+substrate+of+caspase-3/-7+and+an+executioner+of+pyroptosis.">Duck gasdermin E is a substrate of caspase-3/-7 and an executioner of pyroptosis.</a> Front Immunol. 13, 1078526 (2022).</li><li>M&#252;ller, H., Islam, M. R., Raue, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+on+infectious+bursal+disease-the+past,+the+present+and+the+future.">Research on infectious bursal disease-the past, the present and the future.</a> Vet Microbiol. 97 (1), 153-165 (2003).</li><li>M&#252;ller, H., Scholtissek, C., Becht, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+of+infectious+bursal+disease+virus+consists+of+two+segments+of+double-stranded+RNA.">The genome of infectious bursal disease virus consists of two segments of double-stranded RNA.</a> J Virol. 31 (3), 584-589 (1979).</li><li>Cubas-Gaona, L. L., Diaz-Beneitez, E., Ciscar, M., Rodr&#237;guez, J. F., Rodr&#237;guez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exacerbated+apoptosis+of+cells+infected+with+infectious+bursal+disease+virus+upon+exposure+to+interferon+alpha.">Exacerbated apoptosis of cells infected with infectious bursal disease virus upon exposure to interferon alpha.</a> J Virol. 92 (11), (2018).</li><li>Duan, X., 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=Epigenetic+upregulation+of+chicken+microRNA-16-5p+expression+in+DF-1+cells+following+infection+with+infectious+bursal+disease+virus+(IBDV)+enhances+IBDV-induced+apoptosis+and+viral+replication.">Epigenetic upregulation of chicken microRNA-16-5p expression in DF-1 cells following infection with infectious bursal disease virus (IBDV) enhances IBDV-induced apoptosis and viral replication.</a> J Virol. 94 (2), e01724 (2020).</li><li>Li, Z., 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=Critical+role+for+voltage-dependent+anion+channel+2+in+infectious+bursal+disease+virus-induced+apoptosis+in+host+cells+via+interaction+with+VP5.">Critical role for voltage-dependent anion channel 2 in infectious bursal disease virus-induced apoptosis in host cells via interaction with VP5.</a> J Virol. 86 (3), 1328-1338 (2012).</li><li>Rodr&#237;guez-Lecompte, J. C., Ni&#241;o-Fong, R., Lopez, A., Frederick Markham, R. J., Kibenge, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+bursal+disease+virus+(IBDV)+induces+apoptosis+in+chicken+B+cells.">Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells.</a> Comp Immunol Microbiol Infect Dis. 28 (4), 321-337 (2005).</li><li>Wang, S., Liu, Y., Zhang, L., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+monitoring+cancer+cell+pyroptosis.">Methods for monitoring cancer cell pyroptosis.</a> Cancer Biol Med. 19 (4), 398-414 (2021).</li><li>Zhang, Y., Chen, X., Gueydan, C., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+changes+during+programmed+cell+deaths.">Plasma membrane changes during programmed cell deaths.</a> Cell Research. 28 (1), 9-21 (2018).</li><li>Chen, X., 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=Pyroptosis+is+driven+by+non-selective+gasdermin-d+pore+and+its+morphology+is+different+from+mlkl+channel-mediated+necroptosis.">Pyroptosis is driven by non-selective gasdermin-d pore and its morphology is different from mlkl channel-mediated necroptosis.</a> Cell Research. 26 (9), 1007-1020 (2016).</li></ol>

作者没有什么可披露的。

本文介绍了一种使用流式细胞术检查焦亡的有效方法，该方法通过使用 Alexa Fluor 647 标记的抗 chGSDME-NT MCAB 和 PI 对感染细胞进行双重染色来实现。这种方法也可以应用于各种细胞类型，以区分焦亡与其他类型的细胞死亡，例如细胞凋亡和坏死。
焦亡是一种炎症类型的程序性细胞死亡，主要依赖于哺乳动物中 Gasdermin （GSDM） D 诱导的质膜孔形成</sup...

细胞焦亡是一种炎症类型的程序性细胞死亡，主要取决于哺乳动物体内 Gasdermin （GSDM） D 形成质膜孔 1,2,3。由于鸡 GSDMD 的遗传缺陷 4,5，鸡焦亡的机制仍然难以捉摸。Gasdermin 家族包含保守蛋白，包括 GSDMA、GSDMB、GSDMC、GSDMD、GSDME 和 DFNB59 3,6。研究报道，来自硬骨鱼和鸭的 GSDME 被 caspase-1/3/7 或 caspase-3/7 裂解，以诱导细胞焦亡 7,8。然而，GSDME 介导的焦亡在宿主对鸡病原感染的反应中的作用仍有待阐明。
传染性法氏囊病 （IBD） 是一种由 IBDV9 引起的急性、高度传染性和免疫抑制性的家禽疾病。IBDV 是一种无包膜的双节段双链 （ds） RNA 病毒，属于 Birnaviridae 科的 Avibirnavirus 属10。其他人和我们实验室先前的研究表明，IBDV 感染通过不同的途径诱导宿主细胞死亡11、12、13、14。先前的发现表明，IBDV 感染会触发乳酸脱氢酶 （LDH） 的释放，乳酸脱氢酶 （LDH） 是裂解细胞死亡的指标 6,15，表明 IBDV 感染会诱导宿主细胞的裂解细胞死亡。此外，数据显示 IBDV 感染的细胞表现出细胞焦亡的形态学特征，包括细胞肿胀，质膜吹出大气泡和碘化丙啶 （PI） 染色阳性，表明 IBDV 感染诱导细胞焦亡。
考虑到裂解的 GSDM 的 N 端片段 （GSDM-NT） 在焦亡细胞中形成膜孔是焦亡的标志，理论上，通过使用特异性抗体检查细胞膜上的 GSDM-NT，可以通过流式细胞术检测焦亡细胞。在禽焦亡细胞中，鸡 Gasdermin E （chGSDME-NT） 的 N 端片段形成膜孔，使碘化丙啶 （PI） 能够穿过并结合 DNA。因此，可以使用流式细胞术检测焦亡细胞的比例，从而将焦亡与其他形式的细胞死亡（如细胞凋亡和坏死）区分开来。然而，尚未报道通过流式细胞术检查焦亡细胞的方法。在本研究中，DF-1 细胞感染 IBDV，并使用针对 chGSDME-NT 片段 （膜结合） 的单克隆抗体 （MCAB） 和 PI 染色进行流式细胞术检查焦亡细胞。 令人惊讶的是，流式细胞术有效地检测到了焦亡细胞。此外，可以量化焦亡细胞的比例。这些发现为确定焦亡提供了一种强大而有效的方法。
本文介绍了一种使用抗 chGSDME-NT MCAB 和 PI 染色通过流式细胞术检查 IBDV 感染细胞焦亡的方法。该方法也可以扩展到其他病原体感染的细胞，并应用于检查各种细胞类型的焦亡，将它们与其他形式的细胞死亡区分开来。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL round-bottom polystyrene tube (12 &#215; 75 mm)</td>
			<td>Corning Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 antibody labeling kits</td>
			<td>Thermo Fisher Scientific</td>
			<td>A20186</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-chGSDME-CT McAb</td>
			<td>SAE Biomedical Tech Company (Zhongshan, China)</td>
			<td>EU0228</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-chGSDME-NT McAb</td>
			<td>SAE Biomedical Tech Company (Zhongshan, China)</td>
			<td>EU0227</td>
			<td></td>
		</tr>
		<tr>
			<td>CellQuest software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic Freezing Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)&#160;</td>
			<td>Gibco by Life Technologies</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F0193-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>FACSCalibur</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Staining Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-4222-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Qiu-jing Biochemical Reagent &#38; Instrument Company (Shanghai, China)</td>
			<td>YX-JSB52</td>
			<td></td>
		</tr>
		<tr>
			<td>IBDV Lx strain</td>
			<td></td>
			<td></td>
			<td>IBDV Lx strain was kindly provided by Dr. Jue Liu, Beijing Academy of Agriculture and Forestry, Beijing, China</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Chongqing Photoelectric Instrument Company</td>
			<td>XDS-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse IgG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>M&#38;C&#160; Gene Technology</td>
			<td>CC017</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide(PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>M&#38;C&#160; Gene Technology</td>
			<td>CC008</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Oscillator</td>
			<td>MIULAB</td>
			<td>MIX-28+</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of pyroptosis by flow cytometry

细胞焦亡是一种炎症类型的程序性细胞死亡，主要由裂解的 Gasdermin （GSDM） 家族蛋白产生的 N 端形成质膜孔驱动。通过 Western Blot 检查膜附着的 GSDM-NT 是评估焦亡的最常用方法。然而，使用这种方法很难区分细胞焦亡和其他形式的细胞死亡。在本研究中，采用传染性法氏囊病病毒 （IBDV） 感染的 DF-1 细胞作为模型，利用针对鸡 GSDME N 端片段 （chGSDME-NT） 的特异性抗体和碘化丙啶 （PI） 染色，通过流式细胞术量化发生焦亡的细胞比例。使用 Alexa Fluor 647 标记的抗 chGSDME-NT 抗体，通过流式细胞术很容易检测到 chGSDME-NT 阳性细胞。此外，IBDV 感染细胞中 chGSDME-NT/PI 双阳性细胞的比例 （约 33%） 显著大于模拟感染对照 （P < 0.001）。这些发现表明，通过流式细胞术检查膜结合的 chGSDME-NT 是确定细胞死亡细胞中焦亡细胞的有效方法。

流式细胞术可以很容易地检测到 IBDV 感染的 DF-1 细胞膜上的 chGSDME-NT 焦亡细胞最重要的特征之一是 Gasdermin 切割产生的 GSDM-NT 片段形成膜孔。因此，理论上可以通过流式细胞术通过使用特异性抗体检查细胞膜上的 GSDM-NT 来检测焦亡细胞。因此，用 IBDV 感染 DF-1 细胞，并使用 Alexa Fluor 647 标记的抗 chGSDME-NT MCAB 和 PI 染色通过流式细胞术检查焦亡细胞。<sup...

作者聚焦：流式细胞术测定禽细胞焦亡

本文介绍了使用针对鸡 GSDME 的 N 端片段 （chGSDME-NT） 和碘化丙啶 （PI） 的抗体进行双重染色后，使用流式细胞术鉴定焦亡细胞。

流式细胞术检查细胞焦亡

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated ...

examination-of-pyroptosis-by-flow-cytometry

Research

JoVE Journal

Immunology and Infection

Examination of Pyroptosis by Flow Cytometry

831 次观看.  National Key Laboratory of Veterinary Public Health Security. 我们研究团队的目标是确定禽细胞是否经历类似于哺乳动物细胞的焦亡。此外，我们的目标是通过更准确、更轻松的方法确定焦亡。禽类物种中的许多基因与哺乳动物中的基因显著不同，因此很难将哺乳动物细胞研究中常用的试剂用于禽类细胞死亡研究。因此，对禽细胞死亡的研究比对人或小鼠细胞的研究要困难得多。我们利用由 gasdermin N 末?...

视频: 作者聚焦：流式细胞术测定禽细胞焦亡

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...