Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry
Tóm tắt
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 (HO342) 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 HO342 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‐G1” peak. The HO342/7AAD 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. © 1994 Wiley‐Liss, Inc.
Từ khóa
Tài liệu tham khảo
Boyum A, 1968, Isolation of mononuclear cells and granulocytes from human blood, Scand J Clin Invest, 97, 77
Gorczyca W, 1993, Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays, Cancer Res, 53, 1945
Gougeon ML, 1992, Immunodeficiency in HIV Infection and AIDS, 115
Kubbies M, 1993, Flow cytometric analysis of cell death pattern of apoptosis and necrosis, Cytometry, 14, 14
Li J, 1992, Differential response of human thymus cells to CD2 antibodies: Fragmentation of DNA of CD45RO+ and proliferation of CD45RO‐ subsets, Immunology, 75, 305
McConkey DJ, 1989, Calcium‐dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex, J Immunol, 143, 1801, 10.4049/jimmunol.143.6.1801
Ormerod MG, 1993, Characterization of apoptosis in rat thymocytes by flow cytometry, Cytometry, 14, 14
Tedder TF, 1985, Human lymphocyte differentiation antigens HB‐10 and HB‐11 I. Ontogeny of expression, J Immunol, 134, 2983, 10.4049/jimmunol.134.5.2983
Uittenbogaart CH, 1983, Growth of human malignant cell lines in serum free medium. In Vitro, 19, 72