A RAPID AND SIMPLE METHOD FOR MEASURING THYMOCYTE APOPTOSIS BY PROPIDIUM IODIDE STAINING AND FLOW-CYTOMETRY

被引:4497
作者
NICOLETTI, I
MIGLIORATI, G
PAGLIACCI, MC
GRIGNANI, F
RICCARDI, C
机构
[1] UNIV PERUGIA,IST CLIN MED 1,I-06100 PERUGIA,ITALY
[2] UNIV PERUGIA,IST FARMACOL,I-06100 PERUGIA,ITALY
关键词
APOPTOSIS; THYMOCYTE; DNA FRAGMENTATION; FLOW CYTOMETRY; DEXAMETHASONE;
D O I
10.1016/0022-1759(91)90198-O
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4-degrees-C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes.
引用
收藏
页码:271 / 279
页数:9
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