Department of Chemical Pathology, Faculty of Health Sciences, University of Pretoria; Tshwane Academic Division, National Health Laboratory Service;
Department of Chemical Pathology, Faculty of Health Sciences, University of Pretoria.
J Vis Exp. 2021 Aug 26(174). doi: 10.3791/62663.
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 °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.
使用台式流式细胞仪研究了放线菌素 D 处理的 SiHa 宫颈癌细胞中的细胞凋亡生物标志物。在实验和对照培养物中测量了细胞凋亡的早期生物标志物（Annexin V 和线粒体膜电位）和晚期生物标志物（caspases 3 和 7 以及 DNA 损伤）。将细胞在 37°C、5%CO2 的加湿培养箱中孵育 24 小时。然后使用胰蛋白酶将细胞分离，并使用流式细胞术细胞计数测定法进行计数。使用 Annexin V 测定法、线粒体电化学跨膜电位测定法、caspase 3/7 测定法和 DNA 损伤测定法进一步分析细胞凋亡。本文概述了细胞凋亡和传统流式细胞术，并详细说明了处理和分析 SiHa 细胞的流式细胞术方案。结果描述了阳性、阴性和次优的实验数据。还讨论了使用这种分析平台进行细胞凋亡流式细胞分析的解释和注意事项。流式细胞术分析为细胞凋亡的早期和晚期生物标志物提供了准确的测量。
