SENSITIVE METHOD FOR MEASURING APOPTOSIS AND CELL-SURFACE PHENOTYPE IN HUMAN THYMOCYTES BY FLOW-CYTOMETRY

被引:196
作者
SCHMID, I
UITTENBOGAART, CH
GIORGI, JV
机构
[1] UNIV CALIF LOS ANGELES, SCH MED, DEPT MED, LOS ANGELES, CA 90024 USA
[2] UNIV CALIF LOS ANGELES, SCH MED, DEPT PEDIAT, LOS ANGELES, CA 90024 USA
[3] UNIV CALIF LOS ANGELES, SCH MED, JONSSON COMPREHENS CANC CTR, LOS ANGELES, CA 90024 USA
来源
CYTOMETRY | 1994年 / 15卷 / 01期
关键词
FLOW CYTOMETRY; APOPTOSIS; HOECHST; 33342; 7-AMINO-ACTINOMYCIN D; HUMAN THYMOCYTES; MULTIPARAMETER ANALYSIS;
D O I
10.1002/cyto.990150104
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A rapid, gentle, and sensitive method for quantification of cells undergoing apoptosis is presented. The method allows the simultaneous determination of dual-color cell surface immunofluorescence. Cells are stained for 7 min with the vital dye Hoechst 33342 (H0342) for identification of live and apoptotic cells. 7-amino-actinomycin D (7-AAD) is added to distinguish cells that have lost membrane integrity from apoptotic and live cells. Due to its spectral properties 7-AAD can be utilized on cells that are dual-surface labelled with fluoresceinisothiocyanate (FITC) and phycoerythrin (PE). The value of the method is demonstrated on human thymocytes, which constitutively undergo programmed cell death and which show an increase in the rate of apoptosis after exposure to the glucocorticoid dexamethasone (DEX). Vital staining with H0342 permits earlier detection of apoptotic changes compared to a staining technique in which cells are treated with a hypotonic citrate solution containing propidium iodide (PI) and the apoptotic cells are represented in a hypodiploid, ''sub-G(1)'' peak. The H0342/7-AAD method may be particularly applicable to studies of programmed cell death in cells in which DNA fragmentation is difficult to detect by decreased DNA stainability. (C) 1994 Wiley-Liss, Inc.
引用
收藏
页码:12 / 20
页数:9
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