Name: characterization of mlkl-mediated plasma membrane rupture in necroptosis
Uploaded: 2018-08-07T15:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

1. 克隆和细胞线生成
<ol><li>PCR 放大 NBB 区域, 对应于氨基酸残滓 1-140 (NBB140), 从人的 MLKL cDNA 的框架标准限制酶的克隆与齐聚的领域 2x FK506 结合蛋白 (2xFKBP 或 2xFV) 和金星荧光蛋白入强力霉素 (Dox) 诱导的逆转录病毒载体 pRetroX-TRE3G 获得 NBB140-2 xfv-金星 (表 1, 图 1 a B)。</li>
	<li>用 MEFs 抗原瞬态转染法从绿色实验室中获得的不朽初级mlkl-/或ripk3- mlkl/ 小鼠胚成纤维细胞 (SV40-large)表达质粒。在 DMEM 周内选择永生化细胞, 并在标准组织中以10% 胎牛血清 (血清)、2毫米 l-谷氨酰胺、100的 U/毫升青霉素和链霉素、1毫米丙酮酸钠和不必要氨基酸在37°c 和 5% CO2中补充。文化孵化器。</li>
	<li>传感器 SV40-immortalized mlkl- MEFs 与反式四环素控制 transactivator (rtTA) 包含的逆转录酶表达的质粒含有 blasticidin 抗性基因 (我们的改良质粒) 和治疗1周与5微克/毫升 blasticidin 选择的细胞稳定表达 rtTA28。</li>
	<li>传感器 rtTA 表达 MEFs 与 Dox 诱导的逆转录病毒载体含有嵌合体 MLKL (pRetroX NBB140-2 xfv-金星普洛), 并选择使用4微克/毫升嘌呤霉素一周, 2 天后转导。</li>
</ol>2. MLKL 介导 Necroptosis 的活体细胞显微成像
<ol><li>板mlkl- MEFs 表示 NBB140-2 xfv-金星密度5万细胞每井 (1 毫升/井) 在24井板, 和孵化过夜 (图 2a)。使用活细胞染色自动计数 (见材料表)。</li>
	<li>用 Dox (0.5 微克/毫升) 治疗细胞12小时, 以诱导 NBB140-2 xfv 金星的表达。</li>
	<li>诱导 necroptosis 与25毫微米 FKBP dimerizer (暗淡) 除25毫微米膜不透水绿色荧光染料 (参见材料表) 在标准 DMEM (参见 1.2)。细胞 necroptosis 积累染料, 因为他们的等离子膜完整性被 NBB140介导的破裂破坏。用三份或四份进行化验。</li>
	<li>使用单或双色成像系统监视 necroptosis (见材料表), 将血管放置在哺乳动物组织培养孵化器中的相应成像单元中。</li>
	<li>打开软件 (参见材料表), 然后在 "任务" 列表下选择选项卡计划即将进行的扫描。 注意:此协议用于使用单色系统进行成像, 但双色系统可使用类似的软件。</li>
	<li>在 "属性" 选项卡中, 选择 "物理布局" 选项卡和 "快速荧光" 中的 "托盘、容器、扫描类型" 和 "扫描模式"。</li>
	<li>通过单击 "时间线" 窗口和获取时间点和频率的总次数 (通常为 6 h 到 12 h, 或每5分钟为快速动力学 necroptosis) 添加 "扫描时间", 并通过选择 "应用程序" 来开始图像获取。"(红色按钮)。</li>
	<li>使用图像分析软件 (参见材料表), 通过从 "任务窗格" (necroptosis) 中选择 "对象计数新的分析, 将固定的分割阈值放在背景水平之上" 来量化, 这可以通过悬停鼠标光标悬停在背景区域的多个图像中使用的分析。</li>
	<li>通过选择使用当前图像预览并根据需要细化阈值级别来检查荧光对象。</li>
	<li>通过打开 "启动新分析" 选项卡, 选择 "时间范围", 选择要分析的井, 输入 "分析作业名称", 然后按 "确定", 将绿色荧光计数集成。</li>
	<li>通过双击相应的作业名称并从图形/导出窗口导出外部绘图软件中的其他分析, Access 从 "分析作业" 选项卡分析数据 (见材料表)。</li>
	<li>定量数据作为绿色荧光正 (+ ve) 计数/毫米2或正常化计数通过汇合并且表达数据作为绿色荧光 + ve 计数或汇合。</li>
	<li>有选择地, 在实验终点, 染色细胞与 100 nM 的膜-permeant 绿色荧光染料 (见材料表)。将数据规范化为% necroptotic 单元格, 作为端点上绿色荧光/绿色荧光的比值。</li>
</ol>3. MLKL 对血浆膜招募和通透的活体细胞共聚焦显微成像
<ol><li>板mlkl- MEFs 表示 NBB140-2 xfv-金星在密度2万个细胞每井 (0.5 毫升/井) 在玻璃底部 4-井显微镜室预处理与50µg/毫升纤维连接蛋白在 PBS 在37°c 为30分钟和孵化为 ~ 12 h (图 2B)。</li>
	<li>用 Dox (0.5 微克/毫升) 治疗细胞12小时, 诱导 NBB140-2 xfv 金星的表达。</li>
	<li>通过孵化与新鲜培养基 (1 毫升/井) 含有 25 nM 暗淡诱导 necroptosis 诱导 NBB140-2 xfv 金星活化和转运到等离子膜。</li>
	<li>将腔室放在旋转圆盘激光扫描共聚焦显微镜上, 安装有环境控制的倒立架和 63 1.4 NA 目标, 并使用选择软件开始图像采集 (见材料表)。</li>
	<li>初始化软件, 选择 "焦点" 图标和 YFP 过滤器配置 (激发波长 515 nm)。</li>
	<li>找到视图和焦点平面的字段, 并使用 "确定焦点" 选项卡参与焦点维护系统。</li>
	<li>要开始获取, 请选择 "捕获" 选项卡, 然后依次分配过滤器配置、适当的 EMCCD 相机曝光时间和增强增益, 以及时间推移采集参数, 包括间隔和采集长度。</li>
	<li>通过使用 515 nm 激光 (影片 1) 的 EMCCD 照相机, 在 necroptosis 期间通过获取图像, 监测荧光 NBB140-2 xfv-金星蛋白的细胞再分布。 注: 漂白是荧光蛋白成像的关注事项。金星是漂白29的抗性荧光蛋白之一。如果使用更容易漂白的荧光蛋白, 图像每三十年代或更长时间, 以允许恢复在成像时间点之间的信号。</li>
	<li>为了监测 NBB140-2 xfv-金星在 necroptosis 期间的等离子膜协会, 每年十年代, 图像在3.3 由全内反射荧光 (TIRF) 显微镜制备, 使用一个装有自动 TIRF 滑块的显微镜和100X 1.4 NA 目标和 EMCCD 照相机 (影片 2)。按照步骤 3.5–3.7, 但根据显微镜室的样品和底部玻璃厚度调整 TIRF 焦点标签内的 TIRF 角。 注: 在 TIRF 分析中, 等离子膜标记 (如 LCK-RFP) 可作为血浆膜定位的控制手段。</li>
	<li>用高斯函数的负归一化二阶导数 (马尔算法) 进行图像处理以提高卷积的质量。</li>
</ol>4. 电子显微术
<ol><li>mlkl MEFs 表达 NBB140-2 xfv-金星密度500万细胞在等离子涂层150毫米细胞培养皿和孵化为 12 h (图 3)。</li>
	<li>用强力霉素 (0.5 微克/毫升) 治疗细胞, 以诱导1-140-2 xfv-金星的表达为 12 h。</li>
	<li>诱导 NBB140-2 xfv-金星活化和易位到血浆膜与 25 nM homodimerizer 5 分钟。这一次是建立在 necroptosis 的动力学中观察到的活细胞成像。</li>
	<li>用10毫升的2.5% 戊二醛、2% 多聚甲醛在0.1 米常使用缓冲液 pH 7.4 (常使用缓冲) 中取出介质并固定细胞, 在37摄氏度前预热。</li>
	<li>收集样品的刮和旋转样品10分钟在 500 x g。放弃上清。</li>
	<li>在0.1 米钠常使用缓冲器中, 用1.5% 钾盐在减少的2% 锇毒气中对样品进行1.5 小时的后缀。锇毒气是一种广泛应用于 TEM 中的染色剂, 可提供对比度。在 necroptosis 细胞扫描电镜前, 我们用 TEM 进行了初步分析。</li>
	<li>将样品在超纯水中冲洗5次, 每 500 x克5 分钟, 然后在自动处理器上用缓冲液 (见 4.6)、水和乙醇漂洗。放弃上清。</li>
	<li>对于扫描电镜, 染色前固定样品与1% 醋酸铀和铅天门冬氨酸重金属染色31。</li>
	<li>通过一系列的酒精和丙烯氧化物溶液来脱水样品。</li>
	<li>将样品嵌入硬树脂32以获得树脂块。</li>
	<li>切厚部分0.5 微米, 以确定正确的分析区域。</li>
	<li>用导电金属 (铱) 的超薄薄膜涂上样品。</li>
	<li>图像和分析样本 (图 3)。为量化, 用电子显微镜扫描5凯文的树脂块表面与暴露的细胞的低放大图像。</li>
	<li>通过扫描电镜或成像软件捕获的单个图像中的细胞可视化, 可以将多个视场连接到一个连续的图像30中。通过在选择软件中生成高分辨率蒙太奇 (见材料表), 大约有100个细胞可以用这种方式来评估 necroptosis 细胞的分数。 注: 扫描电镜评分坏死形态学比较正常细胞特征与前坏死或坏死表型的进展。这些特征包括膜的改变, 包括微绒毛的消失, 扁平化, 细胞破裂从 10 nm 到1µm 的大小, 或失去的器官结构的至少30–40% 的胞浆细胞区。</li>
</ol>5. MLKL 的脂质结合核磁共振 (NMR) 光谱学
<ol><li>用标准蛋白表达和纯化制备15N 标记的 NBB1-156 , 如前所述24。</li>
	<li>溶解500µg 12-二硬脂酸甘油酯锡磷脂酰 3-肌醇磷脂铵盐 (18:0 PI) 和 12-dioleoyl-锡磷脂酰-3-磷-(1 '-肌醇) 铵盐 (18:1 PI) 在500µL 冰冷 CHCl3每个为最后的浓度1毫克/毫升。</li>
	<li>油脂实验1毫克/毫升18:0 和 18:1 PI 在超声波水浴为1分钟在室温或至多5分钟在冰水混合物。</li>
	<li>
整除1毫克/毫升18:0 和 18:1 PI 成清洁玻璃瓶为个别核磁共振滴定系列 (图 4)。用玻璃气密注射器在有机溶剂中转移脂质。
	<ol>
<li>整除132µL 1 毫克/毫升 18:1 PI (摩尔质量 = 880.137 克/摩尔) 在 CHCl3为最后泥沙容量1200µL 在125µM 集中。 注: 为5毫米核磁共振管, 准备连续稀释量 > 1000 µL 和最后核磁共振样品容量 > 500 µL。为3毫米核磁共振管, 准备连续稀释容量 > 300 µL 和最后核磁共振样品容量 > 150 µL。</li>
	</ol>
</li>
	<li>整除1毫克/毫升猪脑 L-α-phosphatidylinositol-4,5-bisphosphate 铵盐 20:9: 1 CHCl3/甲醇/小时2O (脑 PI (45) P2) 成清洁玻璃瓶。</li>
	<li>将小瓶置于氩气气流 (Ar) 或氮气 (N2) 中, 适度流动, 蒸发有机溶剂, 不溅到玻璃玻璃瓶壁上。五到三十分钟通常是足够为10–200µL 有机溶剂的容量。</li>
	<li>用大镊子在 Büchner 或真空瓶底部排列玻璃瓶, 用橡皮塞密封, 并将塑料油管连接到真空线上。夜间在轻度真空下疏散烧瓶, 清除残余有机物。</li>
	<li>覆盖脂膜整除数与惰性气体, 密封与密闭盖子, 并贮存在-20 °c, 用于在一个月或-80 °c, 以长期储存。</li>
	<li>制备含20毫米 naxhypo4 ph 6.8, 10% D2O 和洗涤剂 (+ 炸药) 的核磁共振样品缓冲器, 含20毫米 naxhypo4 ph 6.8, 10% D2o,0.34 毫米的 n-月桂-maltopyranoside。</li>
	<li>在带脂膜的玻璃瓶中加入 + 引爆缓冲器, 达到所需浓度, 在水浴中油脂实验30分钟, 交替超声波10分钟后进行目视检查。 注: 超声波可加热样品。在最终蛋白质样品重组前, 允许冷却至室温15分钟。</li>
	<li>使用1:1 混合物的超声波为所需浓度序列, 对脂洗涤剂胶束进行串联稀释。从125µM 脂浓度开始, 三重复将产生62.5 µM, 31.3 µM, 15.6 µM 脂在0.34 毫米的分布。</li>
	<li>分别制备蛋白质和添加剂的控制和参考核磁共振样品。添加蛋白质和添加剂, 每稀释脂在 + 引爆缓冲。 注: 15N NBB156, 蛋白质添加到最后浓度40µM 和补充2毫米氘 dithiothreitol, 以保持胱氨酸残留减少。</li>
	<li>将适当的样品体积转移到5毫米或3毫米核磁共振管 (见步骤5.4 中的说明)。</li>
	<li>在核磁共振仪器中手动加载样品或在自动化平台 (如 SampleCase 加载器) 中进行排列。</li>
	<li>利用标准脉冲程序获得1H 质子谱的复合核磁共振谱作为质量控制, 其次是 2D 1H-15N 相关谱作为横向松弛优化光谱学 (TROSY) 或异多量子相干性 (SOFAST-HMQC)33。 注: 使用3毫米核磁共振管需要64扫描 1H-15n TROSY (~ 3 小时) 或1h-15n SOFAST-HMQC (~ 90 分钟)。扫描数字为5毫米核磁共振管减半。</li>
	<li>经过处理的2D 核磁共振谱可以添加到卡拉资料库34中, 用于分析或供用户选择的其他软件。</li>
	<li>
通过注释2D 核磁共振共振为每个蛋白质状态的三个良好分散和分辨峰值分析软件。记录包含 NBB156 (图 5A) 的每个缓冲区条件中每个六峰值的峰值振幅。
	<ol>
<li>使用卡拉准备一批集成列表 (单击主菜单标题 "频谱 |安装批次列表 "...)所有的频谱收集和分析使用一个单一的峰值分配文件 (点击主菜单标题 "峰值 |导入 Peaklist "... 并选择用户定义的. 峰值文件, 其峰值为 6, 每个蛋白质状态为 3, 在内定义)。</li>
		<li>调整设置 ("集成商 |调峰模型 "...)X 宽 0.06 ppm 和 Y 宽 0.30 ppm (点击主菜单标题 "积分器 |调峰模型 "...)在将最终输出记录到两个菜单选择之前 (1。单击主菜单标题 "集成商 |集成批次列表 ") (2。单击主菜单标题 "峰值 |导出集成表 ") (图 5B)。 注: M21、V53 和 L105 残留物的骨干酰胺共振已被分配给 NBB156闭支撑状态。采用3D 核磁共振实验24, 将开放支撑状态分配给残余 G130 和 A141 的骨干酰胺共振和 W133 的氢质子侧链共振。</li>
	</ol>
</li>
	<li>通过将参考样品的三个开括号振幅平均, 并计算与这两个参照相关的正常化因子, 使样品直接与不同的磁铁或样品条件进行比较。使用规范化因子计算 NBB156共振的缩放振幅, 并将负振幅设置为零 (表 2)。 示例: 1) 比较不同磁铁的样品: 磁铁 A、NBB156+ 和磁铁 B、NBB156+。2) 洗涤剂比较: 0.34 毫米和1.7 毫米。</li>
	<li>计算每个共振的闭合或开放支撑的比例比例除以最小到最大振幅的所有光谱收集包含适当的参考自由 NBB156和完全开放大括号 NBB156在洗涤剂。</li>
	<li>将规范化的核磁共振振幅作为脂质浓度的函数进行绘制, 并比较 NBB156在不同条件下的比例 (图 5C)。 注: 另外, 对脂质浓度的闭合支撑构象的分数进行分析可以比较脂质结合 NBB156。我们更喜欢前者的分析, 因为它更快, 更好地报告的分数完全参与脂质。</li>
</ol>

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P., 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=RIPK1+blocks+early+postnatal+lethality+mediated+by+caspase-8+and+RIPK3.">RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3.</a> Cell. 157 (5), 1189-1202 (2014).</li><li>Rickard, J. A., 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=RIPK1+regulates+RIPK3-MLKL-driven+systemic+inflammation+and+emergency+hematopoiesis.">RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.</a> Cell. 157 (5), 1175-1188 (2014).</li><li>Kaiser, W. 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=Toll-like+receptor+3-mediated+necrosis+via+TRIF,+RIP3,+and+MLKL.">Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.</a> Journal of Biological Chemistry. 288 (43), 31268-31279 (2013).</li><li>Upton, J. W., Kaiser, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAI+Another+Way:+Necroptotic+Control+of+Viral+Infection.">DAI Another Way: Necroptotic Control of Viral Infection.</a> Cell Host &amp; Microbe. 21 (3), 290-293 (2017).</li><li>Tanzer, M. 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我们提供的技术协议, 我们结合到牵连 MLKL 作为推定刽子手的等离子膜破裂24。除了破译调节 MLKL 介导的 necroptosis 的规管网络外, 这些技术还可以独立用于其他合适的生物系统。实际上, 这些技术是中等到低吞吐量的发现工具。
我们经常使用活细胞成像 NBB140-2 xfv-金星介导 necroptosis, 并诱导其他 MLKL 构造, 机械化解剖 MLKL 的调节 necroptosis 通过诱变和使?...

确定 necroptosis 的遗传成分有助于使用动物模型测试 necroptosis 在生理学和疾病1、2、3、4、5中的含义。RIPK3 或 MLKL 在小鼠中的击倒对发育和成人稳态的影响最小, 表明 necroptosis 对生命3,6不重要。此外, 某些物种不包含 RIPK3 或 MLKL 基因, 支持 necroptosis 在动物7,8的非必要的作用。另一方面, 在实验室诱发的各种病态的击倒动物模型揭示了 necroptosis 在炎症、先天免疫和病毒感染方面的重要作用,9,10,11,12。
Necroptosis 可以通过多种方式激活, 通过不同的先天免疫传感器发出信号, 所有这些都导致激活 RIPK31,13,14。活动 RIPK3 依次 phosphorylates 并激活 MLKL3、4、5、6、7、8、9、10 ,11,12,13,14,15,16,17,18。最受研究的, 也许是最复杂的方式, 导致激活的 RIPK3 涉及死亡受体结扎, 分叉的基础上的信号复合物的下游组成, 以诱导凋亡或 necroptosis1。Necroptosis 发生时, 通过 RIPK1 信号是青睐和结果参与 RIPK319,20。这一结果很容易被青睐的药理抑制或基因删除 caspase 8, 一个假定的内源性抑制剂 necroptosis, 保持 necroptosis 在海湾。RIPK1 绑定并激活 RIPK3。激活 necroptosis 的另一种方法是通过类似于 TLR3/TLR4 信号的像样的受体, 它通过域的适配器诱导的干扰素β (TRIF)21参与并激活 RIPK3。另一种死于 necroptosis 的方法是激活 DNA 传感器戴, 它直接参与并激活 RIPK322。
MLKL 是由一个 n-端螺旋束 (NB) 域和一个 C 终端 pseudokinase 域 (psKD) 组成的胞浆蛋白, 由一个调节支撑区3连接。在正常细胞中, MLKL 被发现在细胞质中, 它被认为是在一个不活跃的复杂与 RIPK314。激活 necroptosis 触发器 RIPK3 磷酸化的 MLKL 在 psKD 的活化回路, 并有可能在 NB 和支撑3,15,23的其他地点。磷酸化诱导 MLKL 的构象变化, 导致从 RIPK314的离解。不清楚的构象变化释放 psKD24的支撑。支撑, 其中包含2螺旋, 介导 MLKL 的齐聚三聚体通过 C 端螺旋25。支撑的 n-端螺旋抑制 NB 域, 这对于膜通透24,26是必不可少的。在分离的情况下, NB 域足以诱发等离子膜通透和 necroptosis16、24、27。在 MLKL (MLKL MEFs) 小鼠胚胎成纤维细胞中, necroptotic 活性为铌的重组。NB 是一种脂质结合领域, 优先从事磷脂磷酸肌醇45磷酸 (PIP2)。我们提出了 MLKL 活化的逐步机制, 其中大括号齐聚通过与 PIP2极性头组24的弱相互作用, 促进了 MLKL 到等离子膜的招募。在膜上, NB 对2的附加高亲和结合点进行了规范的曝光, 在非活动 MLKL 中由大括号屏蔽。总的来说, NB 与 PIP2的多重相互作用破坏了等离子膜的破裂, 尽管这些事件的分子机制尚未被阐明。
在这里, 我们说明了一些具体的方法来描述 MLKL 作为 necroptosis24的刽子手的功能。特别是, 我们专注于最微小的领域 MLKL, NB 和支撑 (NBB), 这是由支撑抑制, 可以通过强制二聚化激活, 以诱导等离子膜破裂和 necroptosis。我们描述了我们的诱导表达系统结合强迫药物诱导 FKBP 介导的二聚化的活细胞成像和电子显微手术细胞 necroptosis。此外, 我们还说明了我们的体外核磁共振分析的相互作用的 NBB 与 phosphatidylinositols (点子)。

<table><tbody><tr><td>&lt;strong&gt;Cloning and cell line generation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pRetroX-TRE3G</td><td>Clontech</td><td>631188</td><td></td></tr><tr><td>Tet-On transactivator plasmid</td><td></td><td></td><td>Llambi &lt;em&gt;et al.&lt;/em&gt;, 2016</td></tr><tr><td>Mouse Embryonic Fibroblasts (MEFs) mlkl-/-</td><td></td><td></td><td>Dillon &lt;em&gt;et al.&lt;/em&gt;, 2014</td></tr><tr><td>Blasticidin S Hydrochloride</td><td>Thermo Fisher Scientific</td><td>BP2647100 CAS#3513-03-9</td><td></td></tr><tr><td>&lt;strong&gt;Cell death quantification and live-cell microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Doxycycline</td><td>Clontech</td><td>631311 CAS# 24390-14-5</td><td></td></tr><tr><td>B/B Homodimerizer AP20187</td><td>Takara</td><td>635059 CAS# 195514-80-8</td><td></td></tr><tr><td>SYTOX Green</td><td>Thermo Fisher Scientific</td><td>S7020</td><td></td></tr><tr><td>Syto16</td><td>Thermo Fisher Scientific</td><td>S7578</td><td></td></tr><tr><td>&lt;strong&gt;NMR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 N Ammonium Chloride</td><td>Cambridge Isotope Laboratories</td><td>NLM-467-10 CAS# 12125-02-9</td><td></td></tr><tr><td>Deuterated DTT</td><td>Cambridge Isotope Laboratories</td><td>DLM-2622-1</td><td></td></tr><tr><td>Deuterium Oxide</td><td>Sigma Aldrich</td><td>617385-1 CAS# 7789-20-0</td><td></td></tr><tr><td>n-Dodecyl-&amp;beta;-D-Maltopyranoside</td><td>Anatrace</td><td>D310 CAS# 69227-93-6</td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt)</td><td>Avanti Polar Lipids</td><td>840046X CAS# 383907-42-4</td><td></td></tr><tr><td>1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (18:0 PI)</td><td>Avanti Polar Lipids</td><td>850143 CAS# 849412-67-5</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phospho-(1&#039;-myo-inositol) (ammonium salt) (18:1)</td><td>Avanti Polar Lipids</td><td>850149 CAS# 799268-53-4</td><td></td></tr><tr><td>&lt;strong&gt;Specialized Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IncuCyte FLR or ZOOM</td><td>Essen BioScience, Inc.</td><td></td><td>Live-cell microscopy imaging</td></tr><tr><td>Helios NanoLab 660 DualBeam&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td></td><td>Electron microscope</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IncuCyte 2011A Rev2 v20111.3.4288 (FLR)</td><td>Essen BioScience, Inc.</td><td>http://www.essenbioscience.com</td><td>Imaging analysis</td></tr><tr><td>FEI MAPS</td><td>Thermo Fisher Scientific</td><td>https://www.fei.com/software/maps/</td><td>EM analysis</td></tr><tr><td>TopSpin v3.2</td><td>Bruker BioSpin</td><td>http://www.bruker.com</td><td>NMR data collection</td></tr><tr><td>CARA v1.9.1.7</td><td></td><td>http://cara.nmr.ch/&amp;nbsp;</td><td>NMR data analysis</td></tr><tr><td>Slidebook</td><td>3i (Intelligent Imaging Innovations)</td><td>https://www.intelligent-imaging.com/slidebook</td><td>Confocal microscopy</td></tr></tbody></table>

characterization of mlkl-mediated plasma membrane rupture in necroptosis

Necroptosis 是由受体相互作用蛋白激酶 3 (RIPK3) 激活触发的程序化细胞死亡通路, phosphorylates 并激活混合谱系激酶样域 pseudokinase, MLKL, 破裂或 permeabilize 等离子膜.Necroptosis 是一种炎症通路, 涉及多种病理, 包括自身免疫、传染性和心血管疾病、中风、变性和癌症。在这里, 我们描述的协议, 可以用来表征 MLKL 作为刽子手的等离子膜破裂在 necroptosis。我们用常规和共聚焦荧光显微学的活细胞成像和固定细胞中的电子显微术来可视化细胞 necroptosis 的过程, 这一起揭示了 MLKL 从细胞质向血浆的再分布膜前诱导大孔的等离子体膜。我们采用体外核磁共振 (NMR) 分析方法, 用脂质来鉴定 MLKL 介导 necroptosis 的调节剂。基于这种方法, 我们确定了定量脂结合偏好和磷脂肌醇磷酸盐 (点子) 作为 MLKL 的关键粘合剂, 这是需要的血浆膜靶向和通透在 necroptosis。

通过最小截断 MLKL 构造, NBB140-2 xfv-金星的诱导表达, 可视化调节 necroptosis 在活细胞中的执行是可能的。该结构保持了诱导等离子膜通透的能力, 并通过 FKBP 盒 (2xFV) 的暗诱导齐聚而活化。我们观察和量化 necroptosis 的活细胞显微成像, 监测动力学 (每5分钟) 摄取的细胞不透水绿色荧光 DNA 结合染料 (图 6a)。这个诱导系统是非常健壮的,...

Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

我们报告的方法, MLKL 介导的等离子膜破裂在 necroptosis 包括常规和共聚焦活细胞显微成像, 扫描电镜, 核磁共振基脂结合。

Necroptosis MLKL 介导的血浆膜破裂特征

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

characterization-mlkl-mediated-plasma-membrane-rupture

Research

JoVE Journal

Biology

10.8K 次观看.  St. Jude Children's Research Hospital. 我们报告的方法, MLKL 介导的等离子膜破裂在 necroptosis 包括常规和共聚焦活细胞显微成像, 扫描电镜, 核磁共振基脂结合。

视频: Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

科研

癌症研究

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.

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

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

免疫与感染

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

神经科学

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

人细胞中HSV-1感染后RIPK3和MLKL的ZBP1依赖性磷酸化的免疫荧光荧光放大的酪胺信号扩增

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical ...

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

评估半胱天冬酶活化以评估先天免疫细胞死亡

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

生物学

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

通过 CLON-G 和 体外 自发死亡测定延长中性粒细胞寿命

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

分析 THP-1 细胞中 LPS/ATP 触发的细胞死亡机制

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

Cell death is a fundamental process in all living organisms. The protocol establishes 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. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show&#160;that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

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

This protocol is based on LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. We use flow cytometry to analyze Annexin V and 7-AAD double staining to detect cell death, using the whole cell and employing scanning electron microscopy to observe the cell membrane morphology.

LPS和ATP诱导的PMA分化THP-1巨噬细胞死亡及其验证

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

Necroptosis MLKL 介导的血浆膜破裂特征

摘要

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此视频中的章节

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

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

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

哺乳动物细胞血浆膜检测效率的高通量测量

人细胞中HSV-1感染后RIPK3和MLKL的ZBP1依赖性磷酸化的免疫荧光荧光放大的酪胺信号扩增

评估半胱天冬酶活化以评估先天免疫细胞死亡

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

通过 CLON-G 和体外自发死亡测定延长中性粒细胞寿命

LPS和ATP诱导的PMA分化THP-1巨噬细胞死亡及其验证

Necroptosis MLKL 介导的血浆膜破裂特征

摘要

探索更多视频

此视频中的章节

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

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

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

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