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><tbody><tr><td>5 mL round-bottom polystyrene tube (12 &amp;times; 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>CO&lt;sub&gt;2&lt;/sub&gt; 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&#039;s Modified Eagle Medium (DMEM)&amp;nbsp;</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 &amp;amp; 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&amp;amp;C&amp;nbsp; 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&amp;amp;C&amp;nbsp; 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

899 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

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

科研

癌症研究

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 ...

免疫与感染

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

放线菌素D处理SiHa宫颈癌细胞中凋亡生物标志物的流式细胞术分析

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

铁死亡髓母细胞瘤细胞中脂质氢过氧化物的 FACS 分析

Revealing the Ferroptotic Phenotype of Medulloblastoma

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named &#34;ferroptosis.&#34; The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic &#34;bubbling&#34; phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

作者聚焦：追踪髓母细胞瘤的铁死亡特征和细胞死亡动力学以用于高级治疗 

Lipid hydroperoxide content represents the most commonly used indicator of ferroptotic cell death. This article demonstrates the step-by-step flow cytometry analysis of lipid hydroperoxide content in cells upon ferroptosis induction.

揭示髓母细胞瘤的铁死亡表型

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and ...

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

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

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

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

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, ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

流式细胞术检查细胞焦亡

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流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

考试流式细胞仪检测胸腺阳性和阴性选择

放线菌素D处理SiHa宫颈癌细胞中凋亡生物标志物的流式细胞术分析

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

揭示髓母细胞瘤的铁死亡表型

揭示髓母细胞瘤的铁死亡表型

揭示髓母细胞瘤的铁死亡表型

评估视网膜小胶质细胞吞噬功能

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测