这项工作得到了国家卫生和医学研究理事会 (GNT1125033 和 GNT1140187) 和澳大利亚研究理事会 (DP170103790) 提供给 i.k.h. P 的赠款的支持。

1. 诱导凋亡
<ol><li>离心细胞样品在 300 x g 5 分钟, 并丢弃上清, 以清除任何预先存在的细胞碎片。 注: 当使用粘附细胞时, 先用1x 磷酸缓冲溶液 (PBS) 在凋亡诱导之前进行种子细胞的冲洗。</li>
	<li>确定单元格编号并收集单元格。 注: 根据化验后的隔离, 我们建议至少 1 x 107细胞的起始细胞数。</li>
	<li>并用重悬在完全媒介 (各自的媒介包含 10% (卷/卷) 胎儿小牛血清, 50 毫升青霉素, 50 µg/毫升链霉素混合物) 为最后浓度 1 x 106细胞/毫升。</li>
	<li>整除 ~ 2 x 106细胞每井6个井板。</li>
	<li>为了诱导细胞凋亡, 用紫外线照射器去除板盖和照射细胞 150 mJ/厘米2 。这大约需要30-60 秒。 注: 在辐照前, 确保 0.5 x 106细胞被保留用于 "未经处理的" 细胞控制。 注: 细胞凋亡也可通过其他方法 (如抗 Fas 或血清饥饿6) 诱发。</li>
	<li>孵育在 37°c, 5% CO2 2-8 小时, 取决于细胞线。</li>
	<li>使用台式上光显微镜, 可视化细胞来确认凋亡形态的存在, 如起泡、apoptopodia 形成和 ApoBD 形成。 注: 40X 放大倍数足够</li>
	<li>使用 P1000 吸管, 吸管和收集凋亡样本。</li>
	<li>用 1x PBS 冲洗盘子, 并与剩余样品相结合, 以确保最大产量。</li>
	<li>收集 ~ 1/10的 ' 整个凋亡样本 ' (是) 控制。</li>
	<li>收集 "未经处理的" 样本。</li>
	<li>离心机和未经处理的样品在 3000 x g 6 分钟。</li>
	<li>并用重悬在1毫升的 1x PBS, 并预留在冰上。</li>
	<li>对于 ApoBD 隔离, 请继续使用其余的凋亡样本执行步骤2或3。</li>
</ol>2. 通过外地资产管制 ApoBD 隔离
<ol><li>将整个样品离心 3000 x 克, 6 分钟。</li>
	<li>去除大部分上清液而不破坏颗粒。</li>
	<li>并用重悬在染色溶液中含有1毫升 1x A5 结合缓冲器, 75 µL A5-FITC, 2 µL 每 TO-PRO-37细胞。</li>
	<li>在黑暗中孵化样品室温10分钟。</li>
	<li>添加1-2 毫升的 1x A5 结合缓冲和离心机样品在 3000 x g 6 分钟, 以消除多余的污渍。 注: 对于混合细胞种群或组织样本, 执行抗体染色步骤, 使用细胞类型特定标记的组合在 1x A5 结合缓冲和孵化在冰上20分钟 (或根据制造商的协议), 然后离心 3000 x g 6分钟.</li>
	<li>并用重悬样品颗粒在3毫升的外地资产管制署缓冲器 (1x PBS, 1x A5 捆绑缓冲器, 10% FSC, 2 毫米 EDTA) 每 1x107细胞。</li>
	<li>过滤通过70µm 细胞过滤器到一个圆底, 聚丙烯 (流式细胞术) 管, 并保持在冰上和在黑暗中的样品。</li>
	<li>打开µm 机并使用100喷嘴执行标准设置, 执行拖放延迟, 并确保稳定的流。</li>
	<li>加载示例并将捕获速度设置为 ~ 1000 事件/秒。</li>
	<li>调整 FSC, SSC, APC (TO-PRO-3) 和 FITC (A5) 电压和位置事件在外地活动地块内, 以确保人口可以清楚地分开。</li>
	<li>记录2万事件。</li>
	<li>在4部分中设置一个门控策略。</li>
	<li>在排序布局中, 将最终的 ApoBD 门添加为所需的排序填充。</li>
	<li>开始获取样本, 并执行测试排序, 收集 500万 ApoBDs 到一个新的管, 其中包含250µL 的运行情况的缓冲区。</li>
	<li>执行系统回冲洗并加载排序的 ApoBDs。</li>
	<li>获取并记录测试排序 ApoBDs。</li>
	<li>通过比较 FSC (y 轴) vs A5 (x 轴事件), 检查 ApoBD 纯度是否为99%。 注: 由于激光漂白而重新分析样品, A5 染色可能略有减少。</li>
	<li>一旦达到高纯度, 加载原始样本并继续排序, 直到获得所需的 ApoBDs 数。 注意: 如果需要, 可以执行双排序以同时隔离 ApoCells 和 ApoBDs。 注: 在长时间排序时, 建议在4摄氏度处孵化收集管。</li>
	<li>一旦排序完成, 收集后排序 ApoBDs 的一小部分, 后排序 ApoCells, 未经处理, 并验证凋亡和确认后排序纯度。 注意: 虽然测试排序和后排序纯度不应该有显著差异, 但这是基于流的设置和稳定性。</li>
</ol>3. 通过差分离心 ApoBD 隔离
<ol><li>将余下的凋亡样本在 300 x g 处离心10分钟。</li>
	<li>收集上清, 留下500µL, 以避免扰乱细胞颗粒, 并添加到一个新的15毫升圆锥管。</li>
	<li>去除剩余的500µL 和并用重悬细胞颗粒在2毫升 1x PBS (这代表了 ' ApoCell 丰富的分数 '。</li>
	<li>将所收集的上清液离心20分钟, 3000克。</li>
	<li>检查颗粒并仔细清除上清 (上清可能含有小的胞外囊泡, 包括微泡和外来体)。</li>
	<li>并用重悬1毫升 1x PBS 中的颗粒 (这代表了 "ApoBD 富集" 分数)</li>
	<li>收集100µL 的每一个可行的, 是, ApoCell 丰富, 并 ApoBD 丰富的样品在一个新的圆底, 聚苯乙烯 (流式细胞仪) 管。</li>
	<li>添加100µL, 其中包含 2x A5 绑定缓冲区、1:100 A5-FITC 和1:1、000 TO-PRO-3</li>
	<li>在室温下在黑暗中孵育10分钟。</li>
	<li>用流式细胞仪分析样品, 采用上述的浇口策略, 验证 ApoBD 的凋亡和纯度的成功诱导。</li>
</ol>4. 流式细胞术门策略
<ol><li>绘制 TO-PRO-3 (y 轴) 反对 FSC (x 轴), 以分离坏死细胞 (TO-PRO-3高) 从所有其他非透事件 (TO-PRO-3低/中级)。</li>
	<li>选择所有非透事件, 并对 A5 绘制 SSC。门的两个人口, 包括人口 1 (P1), SSC中间/高, A5低/中间细胞和人口 2 (P2), 所有其他事件。</li>
	<li>从 P2, 剧情 TO-PRO-3 反对 A5 和选择 A5 中/高事件排除所有细胞碎片。</li>
	<li>选择所有 A5 积极的事件和阴谋 FSC 对 A5。独立的 ApoBDs (fsc低) 从 ApoCells (fsc中间/高)。 注意: 当 ApoBD 通过基于 ApoBDs 的方法进行隔离时, 我们建议最后一步, 选择 ApoBDs 并比较 TO-PRO-3 A5 和选择所有事件。这可以确保最终的分拣门使用荧光而不是 FSC/SSC 参数。</li>
	<li>对于可行的细胞分析, 选择 P1 并执行两个门控策略之一。对于一般可行的细胞分析, 情节 fsc 对 A5 和选择所有 FSC中间/高细胞, 因此删除剩余的细胞碎片。另外, 为了深入分析, 可行的细胞可以与 A5-早期 ApoCells 分离, TO-PRO-3 对 FSC 进行密谋。选择 TO-PRO-3低, fsc中间/高活性细胞和 TO-PRO-3中间体, FSC中间/高A5-早期 ApoCells。</li>
</ol>

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作者没有什么可透露的。

自二十世纪五十年代早期的描述以来, 细胞凋亡领域已取得显著进展, 成为一个突出的研究领域。尽管有广泛的兴趣和广泛的努力, 但由于缺乏适当的方法, 细胞凋亡的某些方面, 特别是 ApoBDs 的形成, 没有得到很好的研究。这些主要包括通过传统的流式细胞仪 A5/PI 分析和 ApoBD 分离的杂质同时跟踪凋亡进展和 ApoBD 形成的局限性。我们最近制定了解决这些方法缺陷的办法。
我们的...

细胞凋亡是一种精心研究的程序性细胞死亡形式, 需要保持生理平衡, 并清除人体内潜在的有害细胞1。细胞凋亡诱导后, 凋亡细胞 (ApoCells) 可进行一系列形态学改变, 并分解成小的膜状囊泡, 称为 ApoBDs。总的来说, 这个过程被称为凋亡细胞拆卸, 可分为3个不同的步骤, 根据形态学2,3。步骤 1 (等离子膜起泡) 的特点是形成的气球状结构在细胞表面称为泡4,5。步骤 2 (凋亡突起形成) 包括长膜突起的形成, 如 apoptopodia, 串珠状 apoptopodia 和微管尖峰6,7,8。最后, 步骤 3 (ApoBD 形成) 包括凋亡突起和/或 ApoCells 的碎片生成 ApoBDs6,9。先前的研究结果表明, ApoBDs 在帮助细胞凋亡和调节细胞间沟通方面起着重要作用。例如, 建议将 ApoCell 的碎片分解为 ApoBDs 可以生成小的 "咬大小" 的碎片, 可以很容易地被周围的吞噬2,10,11。此外, ApoBDs 可能会窝藏一系列生物分子, 如 DNA、RNA 和蛋白质, 这些生物大分子可能被贩运到周围细胞, 以促进细胞通讯12、13、14。为了在功能上调查这些过程, 必须确认三关键参数, 包括 (i) 验证凋亡诱导和 ApoBD 形成, (ii) 分离 ApoBDs, (iii) 确认 ApoBD 纯度。
以前, 许多方法, 包括流式细胞术和电子显微镜已经被用来研究细胞凋亡和 ApoBDs15,16,17,18。然而, ApoBD 检测和量化往往是困难或忽视。例如, 最常规使用的流式细胞术细胞凋亡试验采用蛋白 V (A5, 一种结合外部 "吃-我" 信号磷脂 (PtdSer)) 和核酸染色碘碘化物 (PI)19的蛋白质。然而, 通过使用这种普遍的染色组合, 分析假设只有三种类型的细胞子集 (可行细胞, ApoCells 和坏死细胞) 在样本中。此外, 虽然被许多研究人员认为是 "金本位" 的细胞凋亡, 流式细胞仪检测和随后的数据分析往往排除 ApoBDs 通过一个初始浇口步骤选择 FSC/SSC中间高事件。因此, 我们最近开发了一种新的流式细胞仪检测, 使用 A5 和 TO-PRO-3, 另一个核酸染色, 可选择性地占去 caspase 3/7 激活 pannexin 1 (PANX1) 通道7,20。caspase 3 诱导的 PANX1 活化在细胞凋亡早期 PtdSer 暴露之前, TO-PRO-3 差异性染色凋亡和坏死细胞。此外, 这种方法结合我们的新的门控策略包括所有获得的事件在数据分析和结果, 六细胞/粒子子集被确定, 包括: (i) 可行的细胞 (FSC/SSC中间/高, A5低, TO-PRO-3低), (二) A5-早期 ApoCells (FSC/SSC中间/高, A5低, TO-PRO-3中间), (iii) A5+ ApoCells (FSC/SSC中间/高, A5高, TO-PRO-3中间体), (iv) 坏死细胞或晚 ApoCells (FSC/SSC 中/高, A5高, TO-PRO-3高), (v) ApoBDs (FSC/SSC低, A5中间, TO-PRO-3低/中间体) 和 (vi) 碎片 (FSC/SSC低, A5低, TO-PRO-3低)20。我们的方法强调分析所有细胞/粒子子集的重要性, 更重要的是从细胞和碎片20分离 ApoBDs。因此, 该方法证明了一种有效的方法来验证诱导的凋亡和 ApoBD 形成同时。
传统上, ApoBDs 是通过各种不同的离心方法分离出来的, 即 ApoBDs 可以从细胞或其他基于密度的胞外囊中分离出来。然而, 这种离心方法往往受到低 ApoBD 纯度, 缺乏一个量化步骤, 以确认样品纯度, 和/或无法分离细胞类型特定的 ApoBDs17,21,22。因此, 我们最近开发了两种方法, 一种基于资产管制和一种新的差分离心方法, 可以与我们以前建立的流式细胞术方法相结合, 以验证诱导的凋亡和样品纯度23。ApoBD 隔离通过我们的基于生物传感器的方法可以丰富 ApoBDs 高达99% 纯度, 并可以结合各种细胞类型特异抗体, 以隔离 ApoBDs 从混合细胞种群, 组织样本和体液23。此外, 我们经修正的差分离心法证明了一种有效的分离 ApoBDs > 90% 纯度23的方法。
本文详细介绍了验证凋亡诱导的实验过程, 并对 ApoBD 的形成进行了检测和量化。本文还对采用基于流式和差分离心法的 ApoBD 隔离工作流进行了详细的阐述和比较。代表的数据表明, 所描述的方法为未来的 ApoBD 研究提供了一个有效的前沿工具。

<table><tbody><tr><td>Cells e.g. cultured human THP-1 monocytes (clone number: TIB-202)</td><td>ATCC</td><td>-</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Life Technologies</td><td>22400-089</td><td></td></tr><tr><td>Penicillin-streptomycin mixture</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>FSC</td><td>Gibco</td><td>10099-141</td><td></td></tr><tr><td>1x PBS</td><td>-</td><td>-</td><td></td></tr><tr><td>Annexin V FITC</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>TO-PRO-3 iodide</td><td>Life Technologies</td><td>T3605</td><td>TO-PRO-3 may cause skin, eye and respiratory irration. Avoid direct contact.</td></tr><tr><td>10x Annexin V binding buffer</td><td>BD Bioscience</td><td>556454</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>1001710526</td><td></td></tr><tr><td>Centrifuge tube (15 mL)</td><td>Cellstar</td><td>188271</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 mL)</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>Tissue culture incubator (37 &amp;deg;C, 5% CO2)</td><td>-</td><td>-</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>FACS ARIA III Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Canto II Flow cytometer, configured with two lasers for FITC and APC detection</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FACS Diva 6.1.1 software</td><td>BD Bioscience</td><td>-</td><td></td></tr><tr><td>FlowJo 8.8.6 software</td><td>-</td><td>-</td><td></td></tr><tr><td>&amp;nbsp;UV Stratalinker 1800</td><td>Stratagene</td><td>-</td><td></td></tr></tbody></table>

detection and isolation of apoptotic bodies to high purity

凋亡体 (ApoBDs)、微泡和外来体是胞外囊泡家族的关键成员, ApoBDs 是最大的类型之一。有人建议, ApoBDs 可以通过贩运生物分子来帮助细胞清除以及细胞间通讯。传统的 ApoBDs 识别和分离方法往往由于缺乏准确的定量和低样品纯度而受到限制。在这里, 我们描述了一个工作流, 以确认诱导的凋亡, 验证 ApoBD 的形成, 并隔离 ApoBDs 到高纯度。我们还将概述和比较荧光活化细胞分选 (ApoBDs) 和差分离心法分离的方法。此外, 使用以前建立的基于流式细胞仪的染色和分析方法, 可以确定分离 ApoBDs 的纯度。结合上述方法, THP-1 单核细胞凋亡和凋亡细胞的拆卸被诱导和验证, THP-1 单核细胞产生的 ApoBD 被隔离成纯度为97-99%。

使用这里概述的过程, THP-1 单核细胞凋亡被诱导和 ApoBDs 检测和隔离通过一个基于外地资产管制或差异离心方法 (图 1)。首先, 通过紫外线照射诱导细胞凋亡, 并在2-3 小时孵化后采集标本, 起泡、凋亡膜突形成和 ApoBDs6的生成。采用 TO-PRO-3 和 A5-based 流式细胞仪方法, 通过分离存活细胞、坏死细胞、早期 ApoCells、ApoCells、ApoBDs 和碎屑, 确?...

Watch this Scientific Journal Video about Detection and Isolation of Apoptotic Bodies to High Purity at JoVE.com

Detection and Isolation of Apoptotic Bodies to High Purity

利用流式细胞仪或差速离心的工作流来检测、定量和分离凋亡样本中的凋亡体, 以高纯度。

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. ...

detection-and-isolation-of-apoptotic-bodies-to-high-purity

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

10.5K 次观看.  La Trobe University. 利用流式细胞仪或差速离心的工作流来检测、定量和分离凋亡样本中的凋亡体, 以高纯度。

视频: Detection and Isolation of Apoptotic Bodies to High Purity

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

科研

癌症研究

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

神经科学

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 ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

使用荧光测定法或流式细胞术测量半胱天冬酶活性

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

生物化学

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

改性膜联蛋白V /碘化丙啶检测细胞凋亡细胞死亡的准确评估

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

生物学

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 ...

免疫与感染

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 ...

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 ...