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。

Pyroptosis is an inflammatory type of programmed cell death that mainly depends on the formation of plasma membrane pores by Gasdermin (GSDM) D in mammals1,2,3. Due to the genetic deficiency of GSDMD in chickens4,5, the mechanism of pyroptosis in chickens remains elusive. The Gasdermin family comprises conserved proteins, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB593,6. Studies have reported that GSDME from teleost fish and ducks is cleaved by caspase-1/3/7 or caspase-3/7 to induce pyroptotic cell death7,8. However, the role of GSDME-mediated pyroptosis in the host response to pathogenic infections in chickens remains to be elucidated.
Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive poultry disease caused by IBDV9. IBDV, a non-enveloped bi-segmented double-stranded (ds) RNA virus, belongs to the genus Avibirnavirus in the Birnaviridae family10. Previous studies by others and our laboratory have shown that IBDV infection induces cell death in host cells via different pathways11,12,13,14. Previous findings have demonstrated that IBDV infection triggers the release of lactate dehydrogenase (LDH), an indicator of lytic cell death6,15, suggesting that IBDV infection induces lytic cell death in host cells. Furthermore, the data show that IBDV-infected cells exhibit morphological features of pyroptotic cell death, including cell swelling with large bubbles blowing from the plasma membrane and propidium iodide (PI)-staining positive, suggesting that IBDV infection induces pyroptosis in cells.
Considering that the formation of membrane pores in pyroptotic cells by the N-terminal fragment of cleaved GSDM (GSDM-NT) is a hallmark of pyroptosis, theoretically, pyroptotic cells could be detected by flow cytometry by examining GSDM-NT on the cell membrane using specific antibodies. In avian pyroptotic cells, the N-terminal fragment of chicken Gasdermin E (chGSDME-NT) forms membrane pores, allowing propidium iodide (PI) to pass through and bind DNA. Thus, the proportion of pyroptotic cells can be detected using flow cytometry, thereby distinguishing pyroptosis from other forms of cell death, such as apoptosis and necrosis. However, the method for examining pyroptotic cells by flow cytometry has not been reported. In this study, DF-1 cells were infected with IBDV, and flow cytometry was conducted to examine pyroptotic cells using monoclonal antibodies (McAb) against the chGSDME-NT fragment (membrane-bound) and PI staining. Surprisingly, pyroptotic cells were effectively detected by flow cytometry. Furthermore, the proportion of pyroptotic cells could be quantified. These findings provide a powerful and effective means to determine pyroptosis.
This article describes a method for examining pyroptosis in IBDV-infected cells by flow cytometry using anti-chGSDME-NT McAb and PI staining. This method can also be extended to other pathogen-infected cells and applied to examine various cell types with pyroptosis, distinguishing them from other forms of cell death.

作者聚焦：流式细胞术测定禽细胞焦亡

流式细胞术检查细胞焦亡

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...

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。

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

711 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

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P &#60; 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.

This article describes the identification of pyroptotic cells using flow cytometry after dual staining with antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI).