我们非常感谢成都中医药大学创新中药学研究所的孙佳怡和杨陆在流式细胞术方面的帮助，以及西南中医资源国家重点实验室的陈翠萍在扫描电子显微镜方面的帮助。这项工作得到了国家自然科学基金[82104491]、四川省自然科学基金[2023NSFSC0674]和中国博士后科学基金[2021M693789]的支持。

1. 细胞系和细胞培养
<ol><li>在含有 10% 胎牛血清、1% 青霉素 - 链霉素和 0.05 mM β-巯基乙醇的 RPMI-1640 完全培养基中培养人单核细胞系 THP-1。在37°C的5%CO2 湿润空气中培养细胞。每 2-3 天传代培养一次细胞。 注意：THP-1 是悬浮细胞，选择更换一半的培养基进行传代培养。当在光学显微镜下观察许多细胞片段时，在室温下以300× g 离心3分钟。将细胞重悬于预热的新鲜完全细胞培养基中。</li>
	<li>使用对数生长阶段的细胞（在第4-10代中）进行所有实验。当细胞处于良好状态和对数生长阶段时，它们在100x的光学显微镜下是圆形的，明亮的，致密的，没有团块和细胞碎片。</li>
</ol>2. THP-1细胞的分化
<ol><li>制备浓度为 1 mM 的 PMA 储备溶液（将 1 mg PMA 溶解在 1.62 mL 二甲基亚砜中）。用完全细胞培养基稀释PMA储备溶液至终工作浓度为100nM。</li>
	<li>将培养皿中的细胞收集到 10 mL 无菌离心管中。在室温下以 300 x g 离心 3 分钟，用 3 mL 含有 PMA 的完全培养基重悬细胞。</li>
	<li>打开细胞计数仪和计数软件。将 20 μL 细胞悬液移液到细胞计数板上，并将其插入细胞计数板。点击 Preview B1，转动仪器右下角的旋钮，调整焦距，使中心明亮的单元格在软件上轮廓清晰。单击 “计数” 开始计数。</li>
	<li>根据计数结果，使用含有 PMA 的完全培养基将细胞密度调节至 1 x 106 个细胞/mL。在6孔板中接种细胞，在37°C和5%CO2 下孵育板24小时，然后开始进一步处理。</li>
</ol>3. THP-1细胞的处理
<ol><li>制备浓度为 1 mg/mL 的 LPS 储备溶液（将 1 mg LPS 溶解在 1 mL 磷酸盐缓冲盐水 （PBS） 中）。用无血清培养基将 LPS 储备溶液稀释至最终工作浓度 1 μg/mL。制备浓度为500mM的Na2ATP储备溶液（将0.3026g ATP溶解在1mL PBS中）。</li>
	<li>从孔中吸出含有PMA的培养基，并用PBS 1x洗涤。对照组加入无血清培养基，模型组加入含有LPS的无血清培养基。在进行进一步处理之前，将板在37°C + 5%CO2 下孵育6小时。</li>
	<li>6小时后，将ATP储备溶液加入模型组中，至终工作浓度为500nM。在继续之前，将板在37°C + 5%CO2 下孵育45分钟。</li>
</ol>4.流式细胞术分析（方法1）
<ol><li>实验样品的制备<ol><li>根据步骤 2 在 6 孔板中接种和孵育细胞。建立四个不处理的对照组：一个不染色对照，两个单染色对照（每个染色一个）和一个双染色对照。建立一个治疗组，并按照步骤3进行治疗。</li>
		<li>处理后，从孔中吸出所有上清液，并用PBS 2x洗涤。向每个孔中加入 500 μL 0.05% 胰蛋白酶（非 EDTA），并在培养箱中孵育 30 秒。</li>
		<li>向每个孔中加入 1 mL RPMI-1640 完全培养基以停止细胞分离并将细胞收集到 5 mL 管中。在室温下以300× g 离心3分钟。</li>
		<li>加入 1 mL PBS 以重悬细胞。将带有细胞的试管置于37°C的水浴中3分钟。在室温下以300× g 离心所有样品3分钟。</li>
		<li>用 800 μL 蒸馏水将 200 μL 5x 结合缓冲液稀释至 1x 结合缓冲液。加入 100 μL 1x 结合缓冲液以重悬细胞并将细胞转移到 5 mL 管中。</li>
		<li>将 5 μL 膜联蛋白 V-PE 溶液加入一管对照组细胞中 10 分钟，将 5 μL 7-AAD 溶液加入另一管对照组细胞中 5 分钟（单独作为单染色对照）。轻轻涡旋每个试管以混合染料，并在室温下避光孵育样品。向每个试管中加入 400 μL PBS，轻轻涡旋以终止孵育。</li>
		<li>向剩余的试管中加入 5 μL 膜联蛋白 V-PE 溶液，并在室温下孵育 5 分钟。轻轻涡旋每个试管以混合染料，并在室温下避光孵育样品。5 分钟后，在室温下加入 5 μL 7-AAD 溶液 5 分钟。向每个试管中加入 400 μL PBS，轻轻涡旋以终止孵育。</li>
		<li>使用 35 μm 尼龙网过滤细胞悬浮液，该尼龙网是 5 mL 聚苯乙烯圆底管的一部分。将试管放在冰上并等待测试。测试前轻轻混合，然后将其插入仪器中。</li>
	</ol></li>
	<li>流式细胞术<ol><li>使用荧光激活细胞分选流式细胞仪进行检测。打开流式细胞仪数据分析软件，确认检测器和激光器的性能。单击 流式细胞仪 并选择 Fluidics 启动，等待系统准备就绪。通过单击 “流式细胞仪”>“清洁模式”>“去气流通池”来排除流室气泡。</li>
		<li>单击 “实验”，依次选择 “新建文件夹”、“实验”、“标本”、“试管 ”和 “编辑名称”。管子前面的五角形箭头，点亮时变为绿色。</li>
		<li>单击Cytometer FACSCelesta的图标（快捷键图标），选择参数，然后选择FSC，SSC和PE信号。 单击全局工作表的图标（快捷键图标）以建立 PE 直方图。对于本研究中使用的流式细胞仪，使用 PE 在 586 nm 处绘制膜联蛋白 V，并在 X 轴上绘图;PerCP-Cy5.5 在 700 nm 处为 7-AAD，并在 Y 轴上绘制。</li>
		<li>首先将未染色的对照样品插入仪器中。单击 Acquisition Dashboard 的图标（快捷键图标），从所需群体中获取至少 10,000 个事件。在 FSC-A 和 SSC-A 点图上使用浇口 （P1） 排除碎屑。将流量调整为 中速 并 记录数据。调整电压以将细胞群放置在 FSC/SSC 直方图的对角线上 ，然后单击重新启动。将流量调整为 中速 并 记录数据。</li>
		<li>单击 下一个试管，运行Annexin V和7-AAD的单次染色对照以调整补偿。运行剩余的试管并收集数据。</li>
	</ol></li>
</ol>5. SEM成像（方法2）
<ol><li>实验样品的制备<ol><li>根据步骤 2 在 6 孔板中接种和孵育细胞，并根据步骤 3 进行处理。</li>
		<li>处理后，从孔中吸出所有上清液，并用PBS洗涤2次。向每个孔中加入 500 μL 0.05% 胰蛋白酶（非 EDTA），并在培养箱中孵育 30 秒。</li>
		<li>向每个孔中加入 1 mL RPMI-1640 完全培养基以停止细胞分离并将细胞收集到 5 mL 管中。在室温下以300× g 离心3分钟。</li>
		<li>在室温下用500μL电子显微镜固定剂（2.5%戊二醛，100mM磷盐）固定细胞2小时，然后在4°C下过夜。</li>
		<li>配制铬明矾溶液：将1.0 g明胶加入100 mL超纯水中，70°C水浴加热，搅拌均匀。加入0.05g铬明矾，搅拌均匀，用滤纸过滤。将干净的盖玻片在37°C的铬明矾溶液中浸泡2小时，然后在37°C的烤箱中干燥以备后用。 注意：目的是防止实验过程中的细胞脱离。</li>
		<li>从固定溶液中收集细胞。在室温下以300× g 离心3分钟。加入 100 μL PBS 并轻轻吹走细胞。将细胞悬浮液滴到盖玻片上，静置 5 分钟。吸出所有上清液，每次用PBS洗涤3次，每次5分钟。 注意：必须轻柔地进行操作以防止细胞脱离。不要剧烈摇晃，缓慢移动，并沿壁慢慢加入液体。</li>
		<li>加入 500 μL 1% 锇酸固定液 1 小时。吸出所有上清液，每次用PBS洗涤3次，每次5分钟。</li>
		<li>将样品以一系列乙醇（EtOH）浓度（30%，50%，70%，80%，90%，95%，100%，100%）连续脱水15分钟，每种EtOH溶液15分钟。 注意：盖玻片可以在4°C下在100%EtOH中保存数天，但应尽快干燥以避免过度脱水。</li>
		<li>打开临界点干燥器，打开 CO2 罐，将腔室冷却至 10 °C。 当腔室压力为 0 psi 时，迅速用滤纸包裹样品并将其放入腔室中。</li>
		<li>打开进水阀，通过 CO2 观察窗观察，将液态 CO2 水平升高到两条红线之间（不要超过上限），关闭进水阀。将样品浸泡10分钟。同时打开进气阀和排气阀，使 CO2 平衡 1 分钟（液态 CO2 取代 EtOH）。关闭排气阀，将液态 CO2 液位升高到规定位置，关闭进气阀。</li>
		<li>将温度设置为 35 °C，将压力设置为 1250 psi。一旦温度和压力稳定下来，以 100 psi/min 的速度减压。当腔室压力为零时取出样品，然后关闭仪器。</li>
		<li>使用导电胶带将试样安装在 SEM 铝试样支架上。使用溅射镀膜机在样品上覆盖一层 （2-5 nm） 金。</li>
	</ol></li>
	<li>成像<ol><li>使用高分辨率冷场发射扫描电镜进行成像。将 SEM 的加速电压设置为 3 kV。将工作距离设置为 10 毫米。将放大倍数设置为 1,000 倍以观察全景。将放大倍数设置为 5,000 倍和 20,000 倍以定位质膜并对样品进行成像。 注意：通过 SEM 可视化 PMA 分化的 THP-1 巨噬细胞的过程与其他类型的细胞和组织相似，并且取决于所使用的仪器。参考文献14 提供了详细的 SEM 协议和随附的视频。</li>
	</ol></li>
</ol>

分析 THP-1 细胞中 LPS/ATP 触发的细胞死亡机制

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+pyroptosis,+apoptosis+and+necroptosis+to+PANoptosis:+A+mechanistic+compendium+of+programmed+cell+death+pathways.">From pyroptosis, apoptosis and necroptosis to PANoptosis: A mechanistic compendium of programmed cell death pathways.</a> Comput Str Biotechnol J. 19, 4641-4657 (2021).</li><li>Hou, Y., 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=Rhodiola+crenulata+alleviates+hypobaric+hypoxia-induced+brain+injury+by+maintaining+BBB+integrity+and+balancing+energy+metabolism+dysfunction.">Rhodiola crenulata alleviates hypobaric hypoxia-induced brain injury by maintaining BBB integrity and balancing energy metabolism dysfunction.</a> Phytomedicine. 128, 155529 (2024).</li><li>Shuwen, 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=Cholesterol+induction+in+CD8++T+cell+exhaustion+in+colorectal+cancer+via+the+regulation+of+endoplasmic+reticulum-mitochondria+contact+sites.">Cholesterol induction in CD8+ T cell exhaustion in colorectal cancer via the regulation of endoplasmic reticulum-mitochondria contact sites.</a> Cancer Immunol Immunother. 72 (12), 4441-4456 (2023).</li><li>Luo, 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=Cyclophosphamide+induced+intestinal+injury+is+alleviated+by+blocking+the+TLR9/caspase3/GSDME+mediated+intestinal+epithelium+pyroptosis.">Cyclophosphamide induced intestinal injury is alleviated by blocking the TLR9/caspase3/GSDME mediated intestinal epithelium pyroptosis.</a> Int Immunopharmacol. 119, 110244 (2023).</li></ol>

作者没有什么可透露的。

在本文中，使用两种方法检测 LPS 和 ATP 诱导的 PMA 分化 THP-1 巨噬细胞的死亡。采用膜联蛋白V/7-AAD双染色，采用流式细胞术分析整体染色结果。与其他流式细胞术分析一样，设置一组未染色细胞和两组单染色细胞以排除假阳性和假阴性结果。结果表明，LPS/ATP刺激后，几种细胞失去了膜的完整性，表明可能已经发生了细胞死亡。这种检测方法使我们能够评估整个细胞的细胞死亡。值得注意的是，Anne...

细胞死亡是所有生物体的基本生理过程。近年来，大量研究表明，细胞死亡与生物体内的免疫和平衡有关。研究细胞死亡有助于我们更好地了解疾病的发生和发展。已经描述了几种形式的程序性细胞死亡，并且已经确定了这些过程中的一些关键靶点。焦亡、凋亡和坏死性凋亡是参与内部平衡和疾病1 的三种遗传定义的程序性细胞死亡途径。
细胞焦亡的特征是膜孔的形成和细胞内容物的释放。活化的半胱天冬酶或颗粒酶裂解 gasdermins 以分离其 N 端结构域，然后这些结构域寡聚化，与膜结合并形成孔 2,3,4。Gasdermin 孔提供跨细胞膜的非典型分泌通道，导致下游细胞反应，包括内容物释放和离子流入 2,3,4。最终，细胞最终经历质膜破裂和由 ninjurin-15 促进的焦亡裂解。在凋亡中，活化的 Bax 和 Bak 在线粒体外膜上形成寡聚体并释放细胞色素 C，细胞色素 C 受 BCL-2 家族的促凋亡和抗凋亡蛋白、起始蛋白半胱天冬酶（caspase-8、-9 和 -10）和效应半胱天冬酶（caspase-3、-6 和 -7）之间的平衡调节1,6,7.细胞凋亡的形态变化包括膜起泡、细胞收缩、核碎裂、染色质凝聚和凋亡体形成6,8。受体相互作用的丝氨酸/苏氨酸蛋白激酶 1 （RIPK3） 和混合谱系激酶结构域样 （MLKL） 是坏死性凋亡机制1 的两个下游核心成分。RIPK3 募集并磷酸化 MLKL，p-MLKL 寡聚化，与细胞膜结合，引发膜穿孔，引起离子流入，增加细胞内渗透压，最终导致细胞破裂 6,9。Gasdermins 和 MLKL 与质膜结合并分别介导焦亡和坏死性凋亡，而 BAX/BAK 通过与线粒体6 的外膜结合介导细胞凋亡。
尽管每种途径都有特定的机制和结果，但它们会导致细胞膜上的类似变化。在正常情况下，磷脂酰丝氨酸（PS）仅局限于质膜的内小叶中。然而，在细胞焦亡、凋亡和坏死性凋亡的早期阶段，PS会暴露在质膜外。Caspase-3/caspase-7 激活 TMEM16 和 XKR 家族，导致细胞膜不对称并在凋亡过程中使 PS 外化10。Gasdermin D 介导和 MLKL 介导的 Ca2+ 内流导致细胞膜上磷脂双层对称性丧失和 PS 暴露。暴露发生在细胞膜完整性丧失之前11,12。基于这三种类型的程序性细胞死亡膜的相似变化，我们使用流式细胞术分析膜联蛋白 V/7-氨基放线菌素 D （7-AAD） 双重染色以检测细胞死亡。Annexin V 是一种钙依赖性磷脂结合蛋白，对 PS 具有高亲和力，可作为灵敏探针检测质膜表面暴露的 PS13。7-AAD是一种不能穿过整个细胞膜的核酸染色剂。它类似于碘化丙啶（PI），一种常用的核酸染料。它们具有相似的荧光特性，但7-AAD具有更窄的发射光谱，并且对其他检测通道的干扰较小。由于这些相似性，区分焦亡、细胞凋亡和坏死性凋亡是不够的。我们使用流式细胞术来检测全细胞的细胞死亡。第二种方法用于使用扫描电子显微镜（SEM）捕获细胞膜，以观察单个细胞中细胞死亡的可能形式。
我们建立了脂多糖（LPS）和三磷酸腺苷（ATP）诱导的佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）分化的人单核细胞（THP-1）巨噬细胞脂质沉积模型来观察细胞死亡。该协议侧重于观察细胞死亡，而不是研究机制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% pancreatic enzyme solution (excluding EDTA)</td>
			<td>BOSTER Biological Technology co.ltd</td>
			<td>PYG0068</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-Bottom Tube</td>
			<td>CORNING</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL centrifuge tube</td>
			<td>Labgic Technology Co., Ltd.&#160;</td>
			<td>BS-50-M</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate&#160;</td>
			<td>Sorfa Life Science Research Co.,Ltd</td>
			<td>220100</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V-PE/7-AAD apoptosis analysis kit</td>
			<td>Absin (Shanghai) Biological Technology co.ltd</td>
			<td>abs50007</td>
			<td>Annexin V-PE, 7-AAD, 5&#215;Binding buffer, Apoptosis Positive Control Solution</td>
		</tr>
		<tr>
			<td>celculture CO2 incubator</td>
			<td>Esco (Shanghai) Enterprise Development Co., Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture dish, 100 mm</td>
			<td>Sorfa Life Science Research Co.,Ltd</td>
			<td>230301</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer K2 Fluorescent Cell Counter</td>
			<td>Nexcelom Bioscience LLC</td>
			<td>Cellometer K2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer SD100 Counting Chambers</td>
			<td>Nexcelom Bioscience LLC</td>
			<td>CHT4-SD100-002</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge machine</td>
			<td>Hunan Xiangyi Laboratory Instrument Development Co., Ltd</td>
			<td>L530</td>
			<td></td>
		</tr>
		<tr>
			<td>chromium alum&#160;</td>
			<td>Guangdong Wengjiang Chemical Reagent Co., Ltd.</td>
			<td>PA04354</td>
			<td></td>
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		<tr>
			<td>cover glasses, 9 mm</td>
			<td>Labgic Technology Co., Ltd.&#160;</td>
			<td>BS-09-RC</td>
			<td></td>
		</tr>
		<tr>
			<td>critical point dryer</td>
			<td>Quorum Technologies</td>
			<td>K850</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>BOSTER Biological Technology co.ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>electron microscope fixative</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>G1102</td>
			<td>2.5% glutaric dialdehyde, 100 mM phosphorous salts</td>
		</tr>
		<tr>
			<td>electronic balance</td>
			<td>SHIMADZU</td>
			<td>ATX124</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol absolute</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2021033102</td>
			<td></td>
		</tr>
		<tr>
			<td>flow cytometer</td>
			<td>Becton,Dickinson and Company</td>
			<td>FACSCanto <img alt="Equation 1" src="/files/ftp_upload/66831/66831eq01.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
		<tr>
			<td>flow cytometry analysis software</td>
			<td>Becton,Dickinson and Company</td>
			<td>BD FACSDivaTM Software</td>
			<td></td>
		</tr>
		<tr>
			<td>gelatin</td>
			<td>Guangdong Wengjiang Chemical Reagent Co., Ltd.</td>
			<td>PA00256&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>High resolution cold field emission scanning electron microscope</td>
			<td>TITACHI</td>
			<td>Regulus 8100</td>
			<td></td>
		</tr>
		<tr>
			<td>human monocytic cell line THP-1</td>
			<td>Procell Life Science&#38;Technology Co.,Ltd.</td>
			<td>CL0233</td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope&#160;</td>
			<td>Leica Microsystems Co., Ltd</td>
			<td>DMi1</td>
			<td></td>
		</tr>
		<tr>
			<td>IR Vortex Mixer</td>
			<td>VELP Scientifica Srl</td>
			<td>ZX4</td>
			<td></td>
		</tr>
		<tr>
			<td>lipopolysaccharide&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>L8880</td>
			<td>LPS is derived from Escherichia coli 055:B5</td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>A8270</td>
			<td></td>
		</tr>
		<tr>
			<td>phorbol-12-myristate-13-acetate&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>P6741</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate-buffered saline</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>G4202</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf AG</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette tips, 10 &#956;L</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>T-10PL</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette tips, 1 mL&#160;</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>T-1250L</td>
			<td></td>
		</tr>
		<tr>
			<td>pipette tips, 200 &#956;L</td>
			<td>Servicebio Technology co.ltd&#160;</td>
			<td>T-200L</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 complete culture media</td>
			<td>Procell Life Science&#38;Technology Co.,Ltd.</td>
			<td>CM0233</td>
			<td>RPMI-1640 + 10% FBS + 0.05mM &#946;-mercaptoethanol + 1% P/S</td>
		</tr>
		<tr>
			<td>RPMI-1640 culture media&#160;</td>
			<td>Shanghai&#160;BasalMedia&#160;Technologies Co., LTD.</td>
			<td>K211104</td>
			<td></td>
		</tr>
		<tr>
			<td>sheath fluid</td>
			<td>BECKMAN COULTER</td>
			<td>8546733</td>
			<td></td>
		</tr>
		<tr>
			<td>sputter coater</td>
			<td>Cressington Scientific Instruments Ltd</td>
			<td>108</td>
			<td></td>
		</tr>
		<tr>
			<td>thermostatic water bath</td>
			<td>GUOHUA Electric Appliance CO.,Ltd&#160;</td>
			<td>HH-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

lps and atp-induced death of pma-differentiated thp-1 macrophages and its validation

细胞死亡是所有生物体的基本过程。该方案建立了脂多糖 （LPS） 和三磷酸腺苷 （ATP） 诱导的佛波醇-12-肉豆蔻酸酯-13-乙酸酯 （PMA） 分化的人单核细胞 （THP-1） 巨噬细胞中的脂质沉积模型，以观察细胞死亡。LPS联合ATP是一种经典的炎症诱导方法，常用于研究焦亡，但细胞凋亡和坏死性凋亡也对LPS/ATP的刺激有反应。在正常情况下，磷脂酰丝氨酸仅局限于质膜的内小叶中。然而，在细胞焦亡、凋亡和坏死性凋亡的早期阶段，细胞膜保持完整并暴露于磷脂酰丝氨酸，而在后期阶段，细胞膜失去其完整性。本文采用流式细胞术分析膜联蛋白V和7-氨基放线菌素D（AAD）双染色，检测全细胞的细胞死亡情况。结果显示，在用LPS/ATP刺激后，大量细胞死亡。使用扫描电子显微镜，我们观察了单个细胞中细胞死亡的可能形式。结果表明，在用LPS/ATP刺激后，细胞可能会发生焦亡、凋亡或坏死性凋亡。该协议的重点是观察用LPS / ATP刺激后巨噬细胞的死亡。结果表明，LPS和ATP刺激后的细胞死亡不仅限于焦亡，还可以发生凋亡和坏死性细胞凋亡，有助于研究人员更好地了解LPS和ATP刺激后的细胞死亡，并选择更好的实验方法。

Cell death is a fundamental physiological process in all living organisms. In recent years, substantial studies have shown that cell death is involved in the immunity and balance within the organism. Studying cell death helps us better understand the onset and development of diseases. Several forms of programmed cell death have been described, and some key targets in these processes have been identified. Pyroptosis, apoptosis, and necroptosis are the three genetically defined programmed cell death pathways involved in internal balance and disease1.
Pyroptosis is characterized by the formation of membrane pores and the release of cell contents. Activated caspases or granzymes cleave gasdermins to separate their N-terminal domains, which then oligomerize, bind to the membrane, and form pores2,3,4. The gasdermin pore provides an atypical secretion channel across cellular membranes, resulting in downstream cell responses, including content release and ion influx2,3,4. Ultimately, the cells eventually experience plasma membrane rupture and pyroptotic lysis facilitated by ninjurin-15. In apoptosis, activated Bax and Bak form oligomers on the mitochondrial outer membrane and release cytochrome C, which is regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7)1,6,7. The morphological changes of apoptosis include membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and apoptotic body formation6,8. The receptor-interacting serine/threonine-protein kinase 1 (RIPK3) and mixed-lineage kinase domain-like (MLKL) are two downstream core components of the necroptotic mechanism1. RIPK3 recruits and phosphorylates MLKL, p-MLKL oligomerizes, associates with the cell membrane, initiates membrane perforations, causes ion influx, increases intracellular osmolarity, and eventually cell rupture6,9. Gasdermins and MLKL bind to the plasma membrane and mediate pyroptosis and necroptosis, respectively, while BAX/BAK mediates apoptosis by binding to the outer membrane of mitochondria6.
Although each pathway has specific mechanisms and outcomes, they lead to similar changes on the cell membrane. Under normal circumstances, phosphatidylserine (PS) is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, PS will be exposed, outside of the plasma membrane. Caspase-3/caspase-7 activates TMEM16 and XKR families, which leads to an asymmetrical cell membrane and externalizes&#160;PS during apoptosis10. Gasdermin D-mediated and MLKL-mediated Ca2+ influx leads to loss of the symmetry of the phospholipid bilayer on the cell membrane and the exposure of PS. The exposure occurs before the loss of cell membrane integrity11,12. Based on the similar changes in the membrane of these three types of programmed cell death, we use flow cytometry to analyze Annexin V/7-Aminoactinomycin D (7-AAD) double staining to detect cell death. Annexin V, a calcium-dependent phospholipid-binding protein, has a high affinity for PS, which can serve as a sensitive probe to detect exposed PS on the surface of the plasma membrane13. 7-AAD is a nucleic acid stain that cannot pass through the entire cell membrane. It is similar to propidium iodide (PI), a commonly used nucleic acid dye. They have similar fluorescence characteristics, but 7-AAD has a narrower emission spectrum and less interference with other detection channels. Owing to these similarities, it is not sufficient to distinguish between pyroptosis, apoptosis, and necroptosis. We used flow cytometry to detect the cell death from whole cells. A second method is used to capture the cell membrane using scanning electron microscopy (SEM) to observe the possible forms of cell death in individual cells.
We established a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. This protocol focuses on observing cell death rather than investigating mechanisms.

作者聚焦：THP-1 巨噬细胞对 LPS/ATP 的反应 — 揭示细胞焦亡、细胞凋亡和坏死性凋亡谱

LPS和ATP诱导的PMA分化THP-1巨噬细胞死亡及其验证

We are addressing LPS and ATP-introduced deaths of PMA-differentiated THP-1 macrophages. LPS and ATP-introduced deaths are not limited to pryoptosis but apoptosis and necroptosis can also occur. This will aid in a better understanding of cell death after LPS and ATP stimulation, and choosing more suitable experimental methods.Cryo-EM technology many studies the other structures in tissues, cells, and microorganisms. Cryo-EM provides neo native structure data for diverse molecular complexes. For example, using Cryo-Em techniques to perform 3D reconstruction can help us further understand the characteristic of apoptotic and necroptotic parts.Although pryoptosis is considered dominate in LPS and ATP-introduced cell death, one cannot determine the appropriation of these death forms or whether they all exist. Validating diverse cell death and measuring them will be our area of investigation in the future.

按照方案中的描述处理细胞样品，并进行流式细胞术检测。正常细胞不能用膜联蛋白V和7-AAD（膜联蛋白V-/7-AAD-）染色。在细胞焦亡、凋亡和坏死性凋亡的早期阶段，PS暴露并与Annexin V结合，但细胞膜仍然完整，从细胞外空间排除了7-AAD（Annexin V+/7-AAD-）。在后期阶段，细胞膜失去完整性，细胞同时被膜联蛋白V和7-AAD染色，显示出双重阳性结果（膜联蛋白V+/7-AAD+）。坏死细胞仅用7-AAD（Annexin V-/7-AAD+）...

Read this Scientific Article Protocol about Author Spotlight: THP-1 Macrophage Response to LPS/ATP - Unveiling Pyroptosis, Apoptosis and Necroptosis Spectrum at JoVE.com

该方案基于 LPS 和 ATP 诱导的 PMA 分化 THP-1 巨噬细胞的死亡。我们使用流式细胞术分析膜联蛋白V和7-AAD双染色以检测细胞死亡，使用整个细胞并采用扫描电子显微镜观察细胞膜形态。

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced ...

我们正在解决 LPS 和 ATP 引入的 PMA 分化 THP-1 巨噬细胞死亡问题。LPS 和 ATP 引起的死亡不仅限于肺死亡，而且也可能发生细胞凋亡和坏死性凋亡。这将有助于更好地了解LPS和ATP刺激后的细胞死亡，并选择更合适的实验方法。冷冻电子技术许多研究组织、细胞和微生物中的其他结构。冷冻电子镜为各种分子复合物提供新天然结构数据。例如，使用冷冻电子技术进行 3D 重建可以帮助我们进一步了解凋亡和坏死性凋亡部分的特征。尽管栽亡被认为是LPS和ATP引入的细胞死亡中占主导地位的，但人们无法确定这些死亡形式的挪用性或它们是否都存在。验证多样化的细胞死亡并测量它们将是我们将来的研究领域。

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LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation

1.0K 次观看.  Chengdu University of Traditional Chinese Medicine. 我们正在解决 LPS 和 ATP 引入的 PMA 分化 THP-1 巨噬细胞死亡问题。LPS 和 ATP 引起的死亡不仅限于肺死亡，而且也可能发生细胞凋亡和坏死性凋亡。这将有助于更好地了解LPS和ATP刺激后的细胞死亡，并选择更合适的实验方法。冷冻电子技术许多研究组织、细胞和微生物中的其他结构。冷冻电子镜为各种分子复合物提供新天然结构数据。例如，使用冷冻电子技术进行 3D 重建?...