采用膜联蛋白A5亲和分析法进行细胞死亡的体外测量。

In vitro measurement of cell death with the annexin A5 affinity assay.

作者信息

van Genderen Hugo, Kenis Heidi, Lux Petra, Ungeth Lisette, Maassen Cecile, Deckers Niko, Narula Jagat, Hofstra Leo, Reutelingsperger Chris

机构信息

Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Maastricht, PO Box 616, 6200 MD, Maastricht, The Netherlands.

出版信息

Nat Protoc. 2006;1(1):363-7. doi: 10.1038/nprot.2006.55.

DOI:10.1038/nprot.2006.55

PMID:17406257

Abstract

One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.

摘要

细胞死亡的一个标志是磷脂酰丝氨酸在细胞表面的表达。可以使用与荧光染料偶联的磷脂酰丝氨酸结合蛋白膜联蛋白A5在体外测量细胞表面磷脂酰丝氨酸的表达。这种测量可以通过流式细胞术或共聚焦扫描激光显微镜进行。膜联蛋白A5亲和力测定包括用荧光标记的膜联蛋白A5和碘化丙啶孵育被刺激执行细胞死亡的细胞。活细胞对膜联蛋白A5呈阴性且对碘化丙啶呈阴性，处于细胞死亡早期阶段的细胞对膜联蛋白A5呈阳性且对碘化丙啶呈阴性，继发性坏死细胞对膜联蛋白A5呈阳性且对碘化丙啶呈阳性。流式细胞术整个过程大约需要30分钟，共聚焦扫描激光显微镜则需要45分钟。此处所述的方案中进一步讨论了各种预防措施和注意事项。