This work was supported, in whole or in part, by institutional funds from Dr. Jose&#769; A. Arruda and the Section of Nephrology, University of Illinois at Chicago (to J. S. L.); and funding from the Department of Medicine of McGill University and the Research Institute of the McGill University Health Centre (to J. R.)

1.准备注意:在这个协议中，两个或更多的细胞系，或原代细胞培养，在特定条件下独立地制备。这些制剂包括凋亡的细胞（协议1），坏死细胞（协议2），和健康应答细胞（协议3）。独立制备后，凋亡或坏死的细胞加入到应答细胞，将所得的信号转导被监视（协议4，5和6）。 <ol><li>制备培养基和试剂<ol><li>制备500培养毫升培养基A（使用允许条件下生长的细胞时，请参阅1.2节）通过组合以下:含有4.5g / L葡萄糖，584毫克/升ʟ谷氨酰胺1X Dulbecco氏改良的Eagle氏培养基（DMEM），110毫克/升丙酮酸钠，100单位/ mL青霉素 - 链霉素，10％（体积/体积）胎牛血清（FBS），和10单位/干扰素γ（IFN-γ）的溶液。 </li><li>准备500毫升培养液中的B（使用WH烯非允许的条件下血清饥饿细胞中，看到的是从培养基A.向生长非允许条件（前血清饥饿下细胞）的配方省略FBS和IFN-γ的第1.2节），加10％体积/ v FBS为培养基B. </li><li>制备0.4％（体积/体积）低聚甲醛，pH值7.2，在4％多聚甲醛的库存1X Dulbecco氏磷酸盐缓冲盐水（DPBS）为死细胞的固定。 注意:多聚甲醛是有毒的，并且适当的应谨慎在其使用行使。 </li></ol></li><li>文化BU.MPT细胞<ol><li>从液氮中解冻BU.MPT小区之一小瓶中。 注意:BU.MPT是小鼠肾近端肾小管上皮细胞（PTEC）从转基因小鼠的小鼠主要的控制下轴承SV40大肿瘤抗原（TAG）的温度敏感性（TS）突变（tsA58）衍生的线组织相容性复合体（MHC）H-2K b类I启动子25,26。 </li><li>无菌polystyr板细胞烯真空气体等离子体处理过的组织培养皿（〜每100毫米直径的培养皿5×10 6个细胞）。 </li><li>生长在允许的条件下细胞在湿润的5％（体积/体积） 的 CO 2气氛中。 注意:在允许的条件，其被定义为培养33 - 中的IFN-γ的存在下37ºC，启用tsA58突变标记的转基因的稳定表达。不像小鼠肾脏PTEC的原代培养，其不超过一个单一的通道或两个生存以上，BU.MPT培养下在允许的条件可以无限期地传代。 <ol><li>解冻后传代细胞至少三次实验中的使用之前。 <ol><li>到传代细胞，在37ºC添加1毫升每100mm培养皿0.05％胰蛋白酶，并孵育在湿润的5％（体积/体积） 的 CO 2气氛中进行5分钟。通过加入培养基A.吸液10毫升的分离的细胞中和胰蛋白酶，和分裂1:3至1:5到新100毫米无菌聚苯乙烯真空气体等离子体处理做卷烟UE培养皿。加入介质A，使体积最多可达到每100毫米的菜10毫升。 </li></ol></li></ol></li><li>在实验使用制剂作为应答细胞，生长的细胞在湿润的5％至汇合（体积/体积） 的 CO 2气氛非允许的条件下（ 即在39ºC在不存在的IFN-γ[含10％v培养基B / v FBS]）。 注意:在非允许的条件下，tsA58突变标签转基因的表达是由"95％的抑制，并且BU.MPT细胞行为类似于小鼠肾PTEC的初级培养物。 </li></ol></li></ol> 2.准备凋亡BU.MPT细胞（协议1） 注意:此协议是专用于BU.MPT细胞凋亡细胞的来源，和星形孢菌素的细胞凋亡诱导的方法。可替代地，另一种细胞系，或原代细胞培养中，可使用作为凋亡细胞的来源，与细胞凋亡通过标准化protoco诱导LS对细胞凋亡诱导的特定细胞类型和方法。 <ol><li>通道到100毫米直径的无菌聚苯乙烯真空气体等离子体处理过的组织培养皿后，生长BU.MPT细胞在湿润的5％汇合允许的条件下（体积/体积） 的 CO 2气氛中（ 即在37ºC在培养基A以每100mm培养皿10mL）中，在部分1.2.3中描述。 </li><li>冲洗贴壁细胞单层用培养基B三次，用每​​漂洗10毫升。 </li><li>通过在37ºC在湿润的5％（体积/体积）CO孵育在培养培养基B含有星形孢菌素，非选择性的蛋白激酶抑制剂，所述细胞以1微克/毫升3小时2气氛诱导细胞凋亡。 </li><li>吸出含有星形孢菌素的培养基包含已经从单层脱离"浮动"细胞凋亡。离心机在500 xg离心10分钟该培养基中，弃去上清液，洗涤沉淀用培养基B三次，并添加粒料返回到下一步骤，2.5收集的细胞。 </li><li>通过加入5mM的（乙二胺四乙酸）的EDTA在1×的Ca分离来自步骤2.3和2.4剩余的贴壁细胞2+ -和Mg 2+ -free DPBS以每培养皿1毫升5分钟。吸入包含分离的细胞凋亡的含有EDTA的培养基，并凝聚分离的细胞与来自步骤2.4的"浮动"凋亡细胞在无菌的15毫升的聚苯乙烯离心管中。 </li><li>洗凋亡细胞离心三次，每次500 XG 10分钟和再悬浮在1X 10毫升钙+ 2 -和Mg + 2每洗-免费DPBS。 </li><li>在最后一次洗涤后，悬浮在新鲜培养基B中的凋亡细胞以每毫升〜5×10 6个细胞在使用前，以刺激应答细胞。可替代地，固定洗涤凋亡细胞0.4％（体积/体积）低聚甲醛在1×DPBS 30分钟，然后用培养洗涤三次培养基B，悬浮在培养基B之前</li><li>如适当，确认细胞凋亡的诱导通过流式细胞术使用细胞凋亡的一个单独的制剂（或通过保留一些细胞用于此目的），如前所述6,9,10,15。 注意:早期凋亡细胞具有完整的细胞膜，并且显示为碘化丙啶（PI）阴性和膜联蛋白V阳性细胞减少单元尺寸（相对于活细胞）。晚期凋亡细胞具有非完整细胞膜，并且显示为的PI阳性和膜联蛋白V阳性细胞减少单元尺寸。使用当前协议，典型的制剂含有〜85％早期凋亡和〜15％的晚期凋亡细胞。 </li><li>添加凋亡细胞到BU.MPT应答细胞（协议3）以1凋亡对应答细胞:1的比例，无论是直接或凋亡细胞固定30分钟，用0.4％在1×DPBS（体积/体积）低聚甲醛后。 注:凋亡细胞固定反应的刺激前应不影响的结果，除非所观察到的信号事件是由于释放可溶性介质（ 表1）的。 </li></ol> 3.坏死BU.MPT细胞的制备（协议2） 注意:此协议是专用于BU.MPT细胞坏死细胞的来源，并加热作为坏死诱导的方法。可替代地，另一种细胞系，或原代细胞培养，可以用作坏死细胞的来源，与由为坏死诱导特定细胞类型和方法标准化协议诱导坏死。 <ol><li>通道到100毫米直径的无菌聚苯乙烯真空气体等离子体处理过的组织培养皿后，生长BU.MPT细胞在湿润的5％汇合允许的条件下（体积/体积） 的 CO 2气氛中（ 即在37ºC在培养基A以每盘10毫升），如第1.2.3所述。 </li><li>冲洗贴壁细胞单层用培养基B三次，用每漂洗10毫升。 </li><li>和Mg + 2 -免费DPBS，每道菜1毫升5分钟-加入5毫米EDTA在1X钙+ 2分离的细胞。吸入包含分离的细胞的含EDTA的培养基，并且将细胞悬浮液添加到无菌的15毫升的聚苯乙烯离心管中。 </li><li>洗涤细胞离心三次，每次500 XG 10分钟和再悬浮在10毫升钙+ 2 -和Mg + 2每洗-免费DPBS。 </li><li>在最后一次洗涤后，悬浮在新鲜培养基B中的细胞以每毫升〜5×10 6个细胞。 </li><li>通过在水浴中加热细胞到70ºC45分钟，轻轻涡旋将细胞悬液，每10分钟诱导坏死。 </li><li>孵育2小时的细胞在湿润的5％（体积/体积） 的 CO 2气氛中于37℃。轻轻涡旋细胞悬液每15分钟。 </li><li>适当时，确认坏死的诱导，流式细胞仪使用NE的单独准备crotic细胞（或通过保留一些细胞用于此目的），如前所述6,9,10,15。 注意:坏死细胞已破裂细胞膜，并显示为（相对于活细胞）增加细胞尺寸的PI阳性细胞。使用目前的协议，典型的制剂含有≥95％的坏死细胞。可替代地，坏死和膜完整性的丧失的诱导可以通过台盼蓝染色来确认。 </li></ol> 4.准备BU.MPT响应细胞（协议3） 注意:此协议是专用于BU.MPT细胞作为应答细胞的来源。可替代地，另一种细胞系，或原代细胞培养，可以用作应答细胞。在此之前实验使用，寂灭可以在一夜之间由实验室内的标准化协议引起的。 <ol><li>生长在培养基中一个宽容的条件下，无菌100mm组织培养皿BU.MPT细胞以每盘10毫升，直到他们达到〜85％汇合。 </li><li>吸培养基A，并冲洗细胞培养基B三次，每冲洗10毫升。血清饥饿细胞过夜18至24小时，在湿润的5％非允许的条件下（体积/体积） 的 CO 2气氛中（ 即在以每皿10毫升39ºC在培养基B）中，如在第1.2节中描述4，以诱导平静。可替代地，生长的细胞在培养基中B加10％（V / V）FBS的39ºC一天血清饥饿细胞前在培养介质B过夜无FBS。 </li><li>标签汇合BU.MPT细胞的分离组织培养皿每个实验条件。 </li><li>包括以下六个条件:用15分钟载体或表皮生长因子（EGF）的活BU.MPT应答的刺激，之后他们接触30分钟到或者没有细胞，凋亡细胞，或坏死的细胞。 注意:暴露于死细胞可以刺激或抑制胃肠的活动水平VEN信号分子。刺激上述的低基线最佳检测（ 例如 ，不存在的EGF），而抑制最好由高基线检测（ 例如 ，表皮生长因子的存在下）。因为刺激与死细胞的作用是先验未知的，则建议在不存在和EGF存在两者都执行所有的研究。 </li></ol> 5.凋亡和坏死细胞（协议4）效应器细胞的刺激<ol><li>从静态的预标记的菜肴吸培养基B（血清饥饿）BU.MPT反应细胞（协议3）。 注意:在非许可条件下过夜培养，BU.MPT细胞应该表现得像小鼠肾脏PTEC的小学文化。 </li><li>每100毫米的菜，添加下列之一的2毫升:培养基B（无细胞）;在每毫升〜5×10 6个细胞在培养基B（方案1）的凋亡细胞悬浮液;在或坏死细胞悬浮〜5×10 6每毫升I细胞N培养中B（协议2）。 <ol><li>分发2毫升悬浮死细胞均匀地分布在100mm培养皿，并保持时间他们结到最低限度。为了实现这一目标，分发一滴死细胞悬液滴具有宽尖端吸管上的应答器单元的菜肴的整个表面。用2毫升的体积为最小的稳定时间并避免死细胞的聚集之间的折衷。 注意:使用的悬浮液的死细胞作为刺激蕴涵A号码的特殊的考虑，其更详细地（ 表2和讨论）说明如下。 </li><li>加入2毫升的死细胞以每毫升〜5×10 6个细胞至汇合100mm培养皿，它含有〜10×10 6个细胞1:达到〜1的死对应答细胞的比率。可替代地，为了建立任何观察到的信号事件的剂量依赖性，通过在崇拜渐进稀释连续变化的死应答细胞的比率在协议1和2制备的死细胞悬浮液茜培养基B。 </li></ol></li><li>轻轻转动菜，然后在37ºC在湿润的5％（体积/体积） 的 CO 2气氛中孵育。 </li><li> 30分钟后，刺激应答细胞用载体或50nM的EGF，根据培养皿的预标记。 </li><li>在潮湿的5％（体积/体积） 的 CO 2气氛中孵育在37℃下15分钟。 </li><li>通过加入5毫升冰冷的1X DPBS的，涡旋盘，并且吸取洗涤液洗去死细胞。重复三次。 </li><li>立即将应答细胞菜在冰上进行进一步处理。 </li><li>准备细胞裂解液<ol><li>制备含有以下细胞裂解缓冲液:1×Tris缓冲盐水（TBS）中，10毫焦磷酸钠，0.5％w / v的脱氧胆酸盐，0.1％w / v的SDS，10％甘油，25mM的氟和钠。 <ol><li>细胞裂解，在该IND给出的确切顺序添加以下前夕icated浓度:迷你无EDTA蛋白酶抑制剂混合物（每10毫升的裂解缓冲液的片剂），10％（体积/体积）的Triton X-100，1mM的二硫苏糖醇（DTT），1mM的苯甲基磺酰氟（PMSF），和10 1mM原钒酸钠。 </li></ol></li><li>从步骤5.7添加每冰上新鲜刺激的应答细胞的100mm培养皿冰冷细胞裂解缓冲液的700微升。刮去细胞用细胞刮刀，并传送裂解液预冷1.5mL的微管中。保持在冰上的管15分钟。 </li><li>与20赫兹，每个小于一秒的持续时间的10个脉冲冰超声裂解物。避免在气/液界面的泡沫的产生，因为这可能会过热的样品。完全浸入超声波仪探测到开关上的超声波仪之前含有裂解液的微管，并从裂解物中除去所述探针之前切换超声波仪关闭。 </li><li>离心机以20,000×g离心10分钟，在4℃下，转移上清液至新鲜预冷的微管，并在-70ºC店。 </li><li>执行凝胶电泳和免疫印迹上裂解物的上清液，如前所述6,9,10,15。 </li></ol></li></ol> 6.预防目标和响应之间的直接物理接触使用的半透膜聚碳酸酯（协议5） <ol><li>根据协议3准备BU.MPT应答细胞，但有一个例外;生长的细胞在无菌聚苯乙烯组织培养物处理的12孔簇。 </li><li>在选择的孔，将含有0.4微米聚碳酸酯膜，防止死亡和响应细胞之间的物理相互作用可渗透支持系统。包括对照孔而不在同一实验中的膜。 </li><li>从血清饥饿BU.MPT反应细胞的实验孔吸出物培养介质B。 </li><li>遵循协议4为应答细胞与细胞凋亡或坏死的细胞，用下列修改的刺激: <ol><li>每孔的12井群，添加下列之一的0.1毫升:培养基B（无细胞）;在每毫升〜5×10 6个细胞在培养基B（方案1）的凋亡细胞悬浮液;或以每毫升〜5×10 6个细胞在培养基B（协议2）坏死细胞悬浮液。 注意:作为一个12孔簇的汇合孔含有〜0.5×10 6个细胞，加入0.1毫升的死细胞中的〜 每毫升5×10 6个细胞产生的死对应答细胞的比率〜1:1。 </li><li>到包含可渗透支撑系统井，添加在透气性支承系统内的0.4微米的聚碳酸酯膜的顶部的死细胞，所以死和应答细胞之间没有直接的物理相互作用。 </li></ol></li></ol> 7.抑制吞噬作用与细胞松弛素D（协议6） <ol><li>根据协议3准备BU.MPT反应细胞。 </li><li>在准备的dimeth骨架抑制剂松弛素D基砜（DMSO），在4毫克/毫升（1,000×）。添加BU.MPT应答细胞的标记的前向碗碟溶媒的10微升（DMSO）或细胞松弛素的D10μL的实现在10mL培养基B的4微克/毫升的最终浓度 注:在通过BU.MPT细胞和巨噬细胞系此浓度细胞松弛素D，吞噬被≥90％9,10抑制。 </li><li>在潮湿的5％（体积/体积） 的 CO 2气氛中孵育在37ºC2小时。 </li><li>按照协议4与凋亡或坏死的细胞应答细胞的刺激。 </li><li>如适当，确认死细胞的吞噬作用的抑制通过流式细胞术使用（用于此目的保留或细胞）死亡，应答细胞的单独制剂，如前所述9,10。 </li></ol>

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The authors declare that they have no competing financial interests.

我们在此提供用于表征下列其暴露于经历凋亡，或其他形式的细胞死亡的细胞的存活细胞诱导胞内信号事件的协议。协议强调由暴露于凋亡细胞可调节活应答细胞内的细胞内信号传导途径的多种机制。这些机制包括:互动（通过对死亡细胞或其棚泡和微泡架桥分子或表面决定，通常被称为凋亡小体）与应答细胞的特异性受体;可溶性介质（甚至他们的外代27）的释放;和吞噬机械的接合。下，其中大量的吞噬作用发生的情况下，要考虑的一个额外的机制是介体，例如微RNA，所述凋亡细胞或其囊泡21内富集的递送。我们的协议的意义，因为相比邻疗法的方法，在于通过其凋亡细胞能响应细胞内的调节信号转导的多重机制的仔细解剖。一个主要的优点是，大多数所需的技术是直接的和标准的细胞培养。 而该协议是专用于BU.MPT细胞，永生化小鼠肾PTEC线，因为这两个死和应答细胞的来源，该协议对其他细胞系或原代细胞培养的适应是直截了当和容易实现的。此外，如在我们以前的出版物，死亡和应答细胞的来源不必是相同9,10,19。尽管我们描述了使用星孢素和热量凋亡和坏死的诱导剂，分别为模式和细胞死亡的触发器也可以有所不同。而在暴露于凋亡细胞BU.MPT应答诱导的信号转导事件基本上独立凋亡诱导的方式，我们的HA已经观察到一些差异9,10。出于这个原因，我们推荐细胞凋亡的几种不同的触发器复制的主要成果。例如，如果高度吞噬细胞，例如巨噬细胞，被用作响应者4,6，或如果反应者和死细胞之间相互作用的持续时间足够长的时间以发生实质性的吞噬作用，那么可以考虑的一种无毒的诱导细胞凋亡，如紫外-或γ-辐射4,9,10，排除毒素的潜在吞噬递送。坏死的诱导替代方法，也可以考虑。虽然我们已获得具有加热和细胞的冻融相同的结果，大量细胞聚集和碎片与冻融发生使它的使用存在问题。 几个简单的策略可以帮助阐明对任何信号事件的具体机制观察暴露于凋亡细胞（ 表1 </STRONG&gt;）。这些策略包括:之前他们除了响应细胞凋亡细胞的固定;凋亡和通过半透膜应答细胞的物理分离;利用从发生凋亡作为刺激细胞超速离心收集条件培养基的;凋亡细胞坏死的细胞或乳胶珠的替代品。与某些警告，如表1中详述的，对所观察到的信号事件的效果能精确定位的负责机构。例如，在神经细胞凋亡的受体介导的识别的情况下，该信号事件应该坚持尽管凋亡细胞的固定，并应与由条件培养基或乳胶珠，以及与物理分离置换凋亡细胞被消除死亡和反应细胞。响应于坏死细胞，信令事件应消除或变得相对定向（ 例如 ，抑制与刺激）。 <p类="jove_content"&gt;使用一个细胞悬浮液的作为刺激，而不是可溶性配体，提出几个固有的和很大程度上不可避免实验障碍。这些问题，以及潜在的措施来限制其干涉意识，是成功实施该协议（ 表2）的关键。大多数的这些问题得到从每个响应细胞凋亡细胞的实验的限制数量。这是相对于可溶性因子。在细胞因子的极低飞摩尔浓度特性即使使用，可溶性配体的数量远远超过应答细胞的数量。在凋亡细胞的情况下，然而，空间考虑排除在大大高于每可行响应单元一个死细胞的比例增加的死细胞。一个非常现实的程度，因此，每死细胞事宜。 有限制死对应答细胞比例可这几个后果导致减少或信号事件的遮蔽。与一个可行的应答细胞的物理相互作用，凋亡细胞必须首先通过悬浮介质的柱沉降。甚至以最小的体积，对细胞的沉降时间可以是明显的，只要10至20分钟。细胞内信号传递活动将因此反应细胞之间进行不协调。因此，应答器和死细胞之间的初始接触不能精确定时的，但稍微广泛的倍以内而下降。用于信令上的时间刻度比为解决需要较短发生的事件，这种缺乏同步的可导致信号，使得它不再从背景区分的遮蔽。甚至延长持续时间的信号，如果没有足够的大小，可以容易受到此问题。 除了这些时间问题，空间的事项也有不利的结果产生影响。半月板的效果，特别是在油井和s的菜商场直径（ 例如 ，聚苯乙烯组织培养物处理的96孔簇），或由太有力的移液生成的可导致死亡的细胞的一个非均匀分布流体的电流，从而产生应答细胞中宴席或饥荒的状态。由于测得的信号与在单个孔或板所有应答细胞的衍生，在死细胞中的捕获变异可能会掩盖较弱的信号。 最后一个问题涉及凋亡细胞群的异质性。甚至与细胞凋亡的一个强有力的触发，如星形孢菌素，死细胞的任何制剂将包含在死亡过程的多个阶段的细胞。这成为相关的，如果响应于凋亡细胞的强度依赖于死亡过程10,14内的阶段。由于在平均每个响应单元遇到只是一个凋亡细胞，异质性在这种情况下可能会导致整个检测到的信号事件的削弱。 表2 </强&gt;提供的主要悬浮相关的问题为它们的部分补救的摘要，以及策略。 有独特的使用死细胞作为刺激相关的几个其他实验的担忧。重要的是，培养条件可能会影响不仅应答细胞，也是死细胞本身。例如，血清蛋白，如果存在的话，可以连接到死亡细胞的表面上，并增强或通过响应细胞抑制其识别。在排除故障的不一致结果，或者，由在文献中报道的不同，应注意，以下面的培养条​​件（对于应答细胞和死细胞）:汇合时，通道数，血清的存在下，血清热灭活的程度，与自然和平静的持续时间。最后，需要注意，细胞死亡是所有细胞培养的必然的方面，并且因此应答细胞持续暴露于死低水平细胞。这种接触可能会影响到死细胞的弹丸实验的反应，因为所研究的非常的信号正在不断诱导。信号调制可以随之而来通过正常的反馈通路，或甚至通过应答细胞的亚群的选择，特别是当诱导的信号传导事件涉及存活或增殖。 总之，我们已经描述了一种协议，用于通过与相邻的死亡或正在死亡的细胞的物理相互作用在活细胞中的诱导胞内信号传导事件的确定。虽然重点主要是信令由死细胞的受体介导的识别诱导事件，该协议还允许信令由巨噬细胞吞噬或可溶性介质从死细胞中释放诱导事件的识别。作为刺激使用死细胞的引入，可以阻碍细胞内信号传导事件的检测几个独特的因素。这些铀意识NIQUE因子，以及由死细胞调节细胞内信号传导的多种机制，是这种日益增长的研究领域内的设计和实验解释关键的。这里描述的协议应理解由死亡或濒临死亡的细胞影响他们生活的邻居都在健康和疾病的机理提供帮助。

细胞凋亡，或调控细胞死亡1，有助于在一个基本的方式组织的维持和发展。看过最简单地说，细胞凋亡证岁，损坏或不损害被淘汰到周围组织2,3多余的细胞。凋亡组织稳态的贡献，但是，是相当多的动态和变化。细胞凋亡垂死采集多个新的活动，既分泌和细胞相关，这使他们能够影响相邻活细胞4-10的功能。早期的研究集中于凋亡细胞以抑制炎症11-16的能力，但凋亡细胞也调节范围广泛的细胞功能，包括诸如生命活动作为生存4,9,10，增殖4,9,10，分化17迁移18和19的增长。而且，这些效果并不限于专业吞噬细胞，巨噬细胞样S，但是延伸到几乎所有类型的细胞和谱系，包括传统的非吞噬细胞，例如上皮细胞和内皮细胞7,9,10,18-21。 相邻活细胞暴露于细胞凋亡的特异性响应取决于涉及两个活应答细胞和凋亡细胞本身多种因素。例如，虽然暴露于凋亡细胞同时抑制小鼠巨噬细胞，肾近端小管上皮细胞（PTECs）的增殖，这两种类型的细胞显着地在他们的生存响应于凋亡细胞4,6,9,10不同。凋亡细胞巨噬细胞促进的生存，但诱发PTECs 4,6,9,10的凋亡。值得注意的是，与凋亡细胞的反应可能响应之间一脉相承的细胞，取决于产地10（ 例如 ，肾PTECs 对乳腺上皮的细胞反应的器官，甚至不同细胞）或激活状态22（ 如中性粒细胞）。相反，凋亡细胞可以在视细胞凋亡的性质非常相同的细胞唤起的响应不同刺激10,23或凋亡10,14的阶段。 鉴于响应于凋亡细胞的多种多样和复杂性，大必须小心在解剖负责任何特定结果的信号转导事件和通路。首先，需要可行和凋亡细胞之间的直接物理相互作用的反应必须从那些由凋亡细胞3,6-10释放或生成可溶性介质引起的分化。如果需要的物理相互作用，然后进一步分化应该执行。信号事件可能取决于结合的凋亡细胞的凋亡细胞，随后独立的吞噬，或巨噬细胞吞噬，独立于特定的受体的受体介导的认可<html> 3-7,9,10,19。在后一种情况下，响应不特定于凋亡细胞，并且可以通过任何吞噬材料4,6被触发。 这些区别的重要性可以再次通过对比巨噬细胞和肾PTECs的反应可以理解。对于这两种类型的细胞，暴露于凋亡细胞改变了促存活激酶Akt的活性。调制是依赖于物理作用，因为响应的分离和细胞凋亡由0.4微米的聚碳酸酯膜废除响应4,9,10,19。然而，PTECs的响应是受体介导的和独立的吞噬作用的，而巨噬细胞的反应是由吞噬4,9,10,19驱动。这个结论是通过以下事实暴露于乳胶珠，中性细胞吞噬的刺激，对在PTECs Akt活性没有影响，但模拟凋亡细胞在巨噬细胞4,9的效果增强。 "&gt;虽然少很好的研究，细胞坏死，或意外的细胞死亡1垂死，还调制附近活细胞的功能3-10,19一样凋亡细胞，坏死细胞通过各种机制，特别是在泄漏发挥它们的作用通过其破裂细胞膜3,5,6,9,24细胞内的内容。很多细胞，包括PTECs和巨噬细胞，具有明显的非竞争受体细胞死去坏死5,9。这些受体参与诱导是信号事件常相反的那些细胞凋亡4-10,19诱导的受体的接合。例如，在PTECs，坏死细胞增加的Akt的磷酸化，而凋亡细胞减少的磷酸化9,10,19。 在这里，我们描述了一种协议，用于识别通过相邻凋亡PTECs的受体介导的识别诱导存活肾PTECs细胞内信号传导事件<sup&gt; 9,10,19。而该协议是专用于称为BU.MPT细胞9,10,19,25,26一个条件永生PTEC细胞系，它很容易适应于处于原点非上皮和/或从大于其它器官的细胞系肾。重要的是，使用细胞凋亡作为细胞刺激造成不存在与可溶性配体的某些固有的实验困难。最重要的是凋亡细胞必须加入以悬浮液，而不是作为一种解决方案。这些困难，以及策略来最小化或规避它们，在协议范围内进行讨论。几乎所有的在协议中描述的技术简单，标准的细胞培养。此协议的优势在于它关注由凋亡细胞应答调节细胞内的信号转导多种机制。这些机制包括经由表面决定簇的结合，或对重桥接分子的特异性受体应的小区，可溶性介质的释放，和/或吞噬机械的接合。坏死细胞被包括在所有的实验中，以确保结果是特定细胞死亡的模式，而不是广义响应于死细胞。细心的方法，因为在这个协议中建议，是对我们不断扩大的影响力正在死亡的细胞上发挥他们的现场的邻居，在健康和疾病的认识至关重要。

<table><tbody><tr><td>&lt;strong&gt;Cell culture materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM, 1x with [4.5 g/L] glucose, &amp;#671;-glutamine &amp;amp; sodium-pyruvate</td><td>Mediatech (Manassas, VA)</td><td>10-013-CV</td><td></td></tr><tr><td>HyClone Fetal Clone II, fetal bovine serum</td><td>GE Healthcare (Logan, UT)</td><td>SH30066.03</td><td>add 10% (v/v)&amp;nbsp; to the culture medium A</td></tr><tr><td>Penicillin-Streptomycin-Glutamine, 100x</td><td>Life Technologies (Grand Island, NY)</td><td>10378-016</td><td>add 100 units/mL to the culture media A and B</td></tr><tr><td>&amp;gamma;-Interferon, mouse recombinant, from &lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>Calbiochem, Millipore (Billerica, MA)</td><td>407303</td><td>add 10 units/mL to the culture medium A</td></tr><tr><td>Falcon polystyrene tissue-culture treated dishes, non-pyrogenic</td><td>Corning (Durham, NC)</td><td>353003</td><td>sterile, 100 mm diameter</td></tr><tr><td>Falcon polystyrene centrifuge tubes, conical bottom</td><td>Corning (Durham, NC)</td><td>352095</td><td>sterile, 15 mL</td></tr><tr><td>Trypsin-EDTA, 0.05%, Gibco</td><td>Fisher (Waltham, MA)</td><td>25300062</td><td>sterile, 100 mL</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals and inhibitors&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Staurosporine, from &lt;em&gt;Streptomyces sp&lt;/em&gt;.</td><td>Sigma (St. Louis, MO)</td><td>S4400</td><td>use at [1 &amp;mu;g/mL] for 3 h to induce apoptosis</td></tr><tr><td>Epidermal growth factor (EGF), [1 mg/mL]</td><td>Millipore (Billerica, MA)</td><td>EA140</td><td>use at 50 nM to stimulate responder cells</td></tr><tr><td>UltraPure, 0.5 M EDTA, pH 8.0</td><td>Life Technologies (Grand Island, NY)</td><td>15575-038</td><td>use 5 mM to detach adherent cells</td></tr><tr><td>Trypan blue solution, 0.4%</td><td>MP Biomedicals (Santa Ana, CA)</td><td>91691049</td><td>for dead cells staining</td></tr><tr><td>Paraformaldehyde Solution, 4% in DPBS</td><td>Affymetrix (Santa Clara, CA)</td><td>19943</td><td>use 0.4% (v/v) in 1x DPBS for cell fixation</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS), 1x, Gibco</td><td>Life Technologies (Grand Island, NY)</td><td>14040133</td><td>stock solution</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS), Ca&lt;sup&gt;2+&lt;/sup&gt;- and Mg&lt;sup&gt;2+&lt;/sup&gt;-free, 1x, Gibco</td><td>Life Technologies (Grand Island, NY)</td><td>14190367</td><td>stock solution</td></tr><tr><td>Cytochalasin D, 4 mM in dimethyl sulfoxide (DMSO), from &lt;em&gt;Zygosporium mansonii&lt;/em&gt;</td><td>Sigma (St. Louis, MO)</td><td>C8273</td><td>use at [4 &amp;mu;g/mL] to inhibit phagocytosis</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma (St. Louis, MO)</td><td>D2650</td><td>solvent for cytochalasin D</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>&lt;strong&gt;Cell lysis buffer ingredients&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris-buffered saline (TBS), 10x, pH 7.4</td><td>BioRad (Hercules, CA)</td><td>1706435</td><td>for cell lysis buffer</td></tr><tr><td>Sodium pyrophosphate</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>221368</td><td>10 mM for cell lysis buffer</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>D6750</td><td>0.5% w/v for cell lysis buffer</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>L3771&amp;nbsp;</td><td>0.1% w/v for cell lysis buffer</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>G5516</td><td>10% for cell lysis buffer</td></tr><tr><td>Sodium fluoride</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>450022</td><td>25 mM for cell lysis buffer</td></tr><tr><td>Complete mini EDTA-free protease inhibitor cocktail tablet</td><td>Roche Diagnostics (Indianapolis, IN)</td><td>4693159001</td><td>for cell lysis buffer, 1 tablet per 10 mL of buffer</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>T8787</td><td>10% for cell lysis buffer</td></tr><tr><td>Dithiothreitol (DTT)</td><td>MP Biomedicals (Santa Ana, CA)</td><td>11DTT00005</td><td>1 mM for cell lysis buffer</td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>MP Biomedicals (Santa Ana, CA)</td><td>4800263</td><td>1 mM for cell lysis buffer</td></tr><tr><td>Sodium orthovandate</td><td>MP Biomedicals (Santa Ana, CA)</td><td>2159664</td><td>10 mM for cell lysis buffer</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sorvall T6000B centrifuge</td><td>Du Pont (Wilmington, DE)</td><td>T6000B</td><td></td></tr><tr><td>Fisher Tissuemat, water bath</td><td>Fisher (Waltham, MA)</td><td></td><td></td></tr><tr><td>Sonic Dismembrator (sonicator), model 100</td><td>Fisher (Waltham, MA)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Miscellaneous&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell counting chamber, Neubauer</td><td>Hawksley (Lancing, Sussex, UK)</td><td>AC2000</td><td></td></tr><tr><td>Annexin V-FITC Apoptosis Detection Kit II</td><td>Millipore (Billerica, MA)</td><td>CBA059</td><td>for PI and Annexin V staining to determine the stage of apoptosis</td></tr><tr><td>Eppendorf microcntrifuge tubes, 1.5 mL</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>T9661</td><td></td></tr><tr><td>Transwell permeable support, 0.4 &amp;mu;m polycarbonate membrane</td><td>Corning (Corning, NY)</td><td>3413</td><td>sterile, polystyrene, tissue culture treated</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phospho-glycogen synthase kinase-3&amp;alpha;/&amp;beta; (GSK-3&amp;alpha;/&amp;beta;) (Ser21/9), polyclonal antibody</td><td>Cell Signaling Technology</td><td>9331</td><td>rabbit polyclonal antibody</td></tr><tr><td>Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antobody ( (14C10) )</td><td>Cell Signaling Technology</td><td>2118</td><td>rabbit monoclonal antibody</td></tr></tbody></table>

identification of intracellular signaling events induced in viable cells by interaction with neighboring cells undergoing apoptotic cell death

细胞凋亡奄奄一息，也被称为调控细胞死亡，获得多种新的活动，使他们能够影响相邻的活细胞的功能。重要的活动，如存活，增殖，生长，分化，是由凋亡细胞所调制的许多细胞功能中。识别并响应凋亡细胞的能力似乎是所有单元的普遍特征，无论谱系或始发的器官。然而，多样性和应对凋亡细胞的任务是非常谨慎的解剖负责任何特定结果的信号事件和途径采取的复杂性。特别是，必须由凋亡细胞可以存活应答细胞内影响细胞内信号通路，包括多种机制区分:受体介导的识别凋亡细胞的，由神经细胞凋亡的可溶性介质的释放和/或接合的吞噬细胞性机械。这里，我们提供了用于鉴定正在追踪其暴露于细胞凋亡诱导存活应答细胞的细胞内信号事件的协议。该协议的主要优点在于它支付由细胞凋亡调节信号反应细胞内事件的机制，解剖的注意。而该协议是专用于一个条件永生鼠标近端肾小管细胞系（BU.MPT细胞），它很容易适应于处于原点非上皮和/或从除肾脏等器官的细胞系。作为刺激使用死细胞的引入，可以阻碍细胞内信号传导事件的检测几个独特的因素。这些问题，以及策略来最小化或规避它们，在协议范围内进行讨论。该协议中的应用应援我们的广泛影响力的死亡或濒临死亡的细胞在他们的现场发挥的邻居扩展知识，无论是在健康和disea本身。

在图1中 ，我们提供的示意性描绘的定时和临界参与凋亡细胞的制备（方案1）（方案2）的步骤和坏死细胞和应答细胞与凋亡和坏死细胞的悬浮液的刺激（协议4）。 在图2中，我们提供了示出凋亡细胞的丝氨酸-苏氨酸激酶糖原合酶激酶-3（GSK-3），GSK-3α和GSK-3β的两种同种型的磷酸化的影响的代表性结果。 GSK-3α/β具有在细胞增殖和存活的调节了突出的作用，以及其在葡萄糖代谢的重要作用。在丝氨酸-21和GSK-3β，GSK-3α的磷酸化丝氨酸-9抑制激酶活性，进而，导致减少的磷酸化和增加的β连环蛋白的稳定化。 β-连环蛋白然后易位至细胞核，在那里它促进已知刺激细胞增殖，抑制细胞凋亡的基因的转录。因此，增加了GSK-3α/β的磷酸化与增加的增殖和存活相关，而降低GSK-3α/β的磷酸化降低增殖和存活有关。 静态BU.MPT应答器暴露于凋亡为GSK-3α/β（ 图2A）的30分钟强烈抑制基底磷酸化的细胞。同样地，前暴露于30分钟凋亡靶强烈抑制随后的表皮生长因子（EGF），GSK-3α/β（ 图2A）的诱导磷酸化。与此相反，BU.MPT应答坏死细胞暴露对GSK-3α/β的任基底或EGF诱导的磷酸化没有影响。 为了防止措施BU.MPT反应和细胞凋亡之间的T物理相互作用，BU.MPT反应在渗透支持系统生长并通过0.4微米的聚碳酸酯膜从细胞凋亡的目标分开。 BU.MPT反应和凋亡细胞之间的物理相互作用的预防废除的凋亡的目标，以抑制GSK-3α/β（ 图2B）的磷酸化的能力。来评价吞噬的作用，我们使用了细胞骨架抑制剂细胞松驰素D钮防止死亡细胞的巨噬细胞吞噬。响应于凋亡细胞GSK-3α/β的磷酸化的抑制发生在没有吞噬（ 图2C）。之前我们已经表明，细胞松弛素D，在所使用的浓度，抑制PTEC和巨噬细胞吞噬经≥90％9,10。两者合计，我们的结果表明，GSK3α/β的磷酸化的细胞凋亡介导的抑制，需要直接的物理相互作用betwe<html>恩目标和应答细胞，但是独立吞噬。 <img alt="图1" src="/files/ftp_upload/54980/54980fig1.jpg" /> 图 1。架构细胞内信号的诱导事件存活反应细胞通过物理相互作用与经历细胞死亡相邻小区。该线图描绘了（A）细胞凋亡（APO）细胞和（B）坏死（ 死灵 ）BU.MPT细胞的细胞悬液的准备时间和关键事件，和可行的BU.MPT应答中的（C）的刺激细胞凋亡或坏死细胞的悬浮液。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54980/54980fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /ftp_upload/54980/54980fig2.jpg"/&gt; 图 2。在BU.MPT应答细胞GSK3α/β在暴露于凋亡细胞的磷酸化的抑制需要直接细胞-细胞相互作用，但是独立吞噬的。血清饥饿BU.MPT应答细胞预处理具有（A，B）的车辆或（C）的细胞松弛素D（4微克/毫升），2小时，然后刺激30分钟无细胞（ - ），细胞凋亡（ 载脂蛋白 ），在死细胞或坏死细胞（NEC）到响应的1细胞:1的比例。然后将应答细胞进行洗涤，并孵育在EGF（50纳米）的不存在或存在另外的15分钟，收获前。凋亡细胞的来源是星形孢菌素处理过的BU.MPT细胞。坏死细胞的来源进行热处理BU.MPT细胞。死细胞和BU.MPT响应者之间的互动要么（A，C）畅通，实现直接死细胞应答物理相互作用，或（B）与在可渗透支撑系统死细胞和应答的分离通过一个0.4微米的聚碳酸酯膜。死细胞和非贴壁应答细胞通过洗涤除去，BU.MPT应答细胞裂解物用抗磷酸化GSK3α/β抗体探测，如图所示。相等的加载被探测总甘油醛-3-磷酸脱氢酶（GAPDH）证实。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54980/54980fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td></td><td> 信令事件承担这一机制的影响 </td><td></td></tr><tr><td> 介入 </td><td> 可溶性调解员 </td><td> 受体介导的识别 </strong&gt;的</td><td> 吞噬作用 </td></tr><tr><td>凋亡细胞固定* </td><td>消除</td><td>坚持</td><td>坚持</td></tr><tr><td>细胞凋亡和细胞应答的物理分离† </td><td>坚持</td><td>消除</td><td>消除</td></tr><tr><td>从发生凋亡的细胞stimulus¶超速离心条件培养基</td><td>坚持</td><td>消除</td><td>消除</td></tr><tr><td>坏死细胞的刺激</td><td>消除</td><td>消除或方向相反的反应（ 例如 ，抑制与刺激） </td><td>坚持</td></tr><tr><td>乳胶珠刺激</td><td>消除</td><td>消除</td><td>坚持</td></tr><tr><td>吞噬的药物抑制</td><td>坚持</td><td>坚持</td><td>消除</td></TR&gt; <tr><td colspan="4"> *预测结果假定固定不改变负责受体介导识别和/或吞噬作用临界表面决定簇。 </td></tr><tr><td colspan="4"> †预测结果意味着两个假设:第一，可溶性介体是小到足以穿过膜的分离凋亡和应答细胞，和第二孔，即任何棚囊泡或微泡，通常称为凋亡小体，过大而通过该膜的孔。一种泡或微泡的作用将通过与细胞凋亡和细胞应答与超离心条件培养基的物理隔离观察到的信号事件缺乏一致性来建议。 </td></tr><tr><td colspan="4"> ¶超速离心（30分钟，20000×g）为需要除去任何凋亡小体或其他棚可能模仿EFFE不溶物完整凋亡细胞的CTS。 </td></tr></tbody></table> 表1。 该机制（S）负责信令活细胞活动暴露于凋亡细胞的识别。提供了几个简单的实验策略的潜在机制（S）负责信号事件之间的区分观察暴露于凋亡细胞。这些机制包括:与特定的受体上的应答器单元中的凋亡细胞的物理相互作用，从凋亡细胞可溶性介质的释放，和应答细胞的吞噬机械的接合。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 实验问题 </td><td> 对于最小化和/或规避现有策略 </td></tr><tr><td>数量有限每响应细胞的凋亡细胞（〜1:1的比例）。 </td><td> 1.增加凋亡细胞的数量，但细胞凋亡对应答细胞的比率应保持&lt;2:1。* </td></tr><tr><td>需要对凋亡细胞的重力依赖性沉降。 </td><td> 1.最小化，其中凋亡细胞悬浮介质的体积。 </td></tr><tr><td></td><td> 2.离心盘或板通过悬浮介质驱动凋亡细胞更迅速。 </td></tr><tr><td>凋亡细胞的分布不均匀。 </td><td> 1.小心地与广泛尖端吸管超过应答细胞菜肴的整个表面分布一滴凋亡细胞悬液滴。 </td></tr><tr><td></td><td> 2.避免培养皿或水井，直径&lt;35mm时，其半月板影响可能导致不均匀分布。 </td></tr><tr><td>凋亡细胞群的异质性。 </td><td> 1.使用强诱导剂凋亡。 </td></tr><tr><td></td><td> 2.诱导后，修复细胞凋亡与多聚甲醛和排序流式细胞仪以获得更均匀的人口。 </td></tr><tr><td colspan="2"> *在凋亡到应答细胞比率&gt; 2:1，凋亡细胞会形成合流地毯和额外的细胞凋亡将不再使用应答细胞直接接触</td></tr></tbody></table> 表2中 。实验问题有关与使用细胞悬浮液作为细胞内信号事件的刺激。与使用的细胞悬浮液作为刺激的相关的几个实验的障碍被列出，以及它们的部分补救策略。

Watch this Scientific Journal Video about Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death at JoVE.com

鉴定活细胞与经历凋亡性细胞死亡的邻近细胞相互作用诱导的细胞内信号转导事件生物学 |视频

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

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 ...

identification-intracellular-signaling-events-induced-viable-cells

Nanyang Technological University

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JoVE Journal

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Biology

Cellular Processes

Cell Signaling

SCIENTISTS IN ACTION

8.6K 次观看.  University of Illinois at Chicago. 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.

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

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

科研

癌症研究

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

高纯度凋亡体的检测与分离

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

生物化学

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 ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

免疫与感染

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

测量CD95死亡诱导信号传导复合物的组成和该复合物中Procaspase-8的处理

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

巨噬细胞吞噬胸腺细胞吞噬的实验分析

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

生物学

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

导论细胞死亡

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

教育

JoVE Science Education

高级生物学

细胞生物学

The Extrinsic Apoptotic Pathway

The extrinsic apoptotic pathway is initiated when extracellular death-inducing signals, such as specific cytokines, activate the death receptors expressed on the cell surface. The immune cells involved in this pathway are natural killer cells (NK cells) and cytotoxic T-lymphocytes. NK cells are critical in innate immune response, while cytotoxic T-lymphocytes are associated with adaptive immune response. These cells recognize specific receptors expressed on the altered cells and activate their apoptotic pathway. The extrinsic apoptotic pathway is vital in removing cancerous cells or virus-infected cells.
Another example of external apoptotic signaling occurs during T-cell development in the thymus to eliminate self-reactive T-cells. T-cells are immune cells that bind foreign macromolecules and particles and target them for destruction by the immune system. In order to prevent the immune system from attacking the body&#39;s own cells, T-cells do not target &#8220;self&#8221; proteins (proteins of the same individual). Immature T-cells, or thymocytes, undergo a screening process to determine whether or not they bind self-proteins. If the T-cell binds to self-proteins, it is signaled for apoptosis, thus preventing an autoimmune response.
<h4>Inhibiting Apoptosis</h4>
Anti-apoptotic regulators are proteins that can inhibit the extrinsic apoptotic pathway in cells. The FLICE inhibitory protein, or c-FLIP is one of the master anti-apoptotic regulators that binds to FADD (Fas-associated death domain protein), and caspases such as caspase-8. This binding prevents the formation of the death-inducing signaling complex (DISC), thus preventing the apoptotic pathway.
Some portion of this text is adapted from <a href="https://openstax.org/books/biology-2e/pages/9-3-response-to-the-signal">Openstax, Biology 2e, Section: 9.3</a>

外源性凋亡途径

The extrinsic apoptotic pathway is initiated when extracellular death-inducing signals, such as specific cytokines, activate the death receptors expressed ...