中华医学杂志（英文版）2003，Vol.116Issue(12)：1923-1929,7.
Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells
Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells
ZHAO Jinshun 赵进顺 1Alexandra Schmid-Kotsas 2Hans-Juergen Gross 2Adolf Gruenert 2Max G. Bachem2
作者信息
- 1. Department of Occupational and Environmental Medicine, Public Health School, Southeast University, Nanjing 210009, China
- 2. Department of Clinical Chemistry and Pathobiochemistry, University Hospital, D-89070 Ulm, Germany
- 折叠
摘要
Abstract
Objective To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.Methods Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient.Results One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis.Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50%-90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or AnnexinV-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type.Conclusions Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2.7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis.关键词
apoptosis/staining method/sensitivity/specificity/oxidative stress/adherent cellKey words
apoptosis/staining method/sensitivity/specificity/oxidative stress/adherent cell分类
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ZHAO Jinshun 赵进顺,Alexandra Schmid-Kotsas,Hans-Juergen Gross,Adolf Gruenert,Max G. Bachem..Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells[J].中华医学杂志（英文版）,2003,116(12):1923-1929,7.
