Identification and quantitation of apoptotic cells following anti‐CD3 activation of murine G0 T cells

Wiley - Tập 14 Số 8 - Trang 883-890 - 1993
Francis J. Chrest1, M Buchholz2, Young Ho Kim2, Taeg‐Kyu Kwon2, Albert A. Nordin2
1Clinical Immunology Section, National Institute on Aging, NIH, Baltimore, Maryland 21224.
2Clinical Immunology Section, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, Maryland 21224

Tóm tắt

AbstractMultiparameter flow cytometry and cell sorting were used to examine the process of apoptosis after activation of murine resting T cells with immobilized anti‐CD3. Activated T cells treated with Hoechst 33342 (HO‐33342) and analyzed by flow cytometry showed two major cell populations of high and low fluorescence. These populations were sorted and the DNA extracted and subjected to electrophoresis. Electrophoresis of DNA extracted from T cells showing a low level of HO‐33342 fluorescence (HO‐Low) resulted in a typical ladder pattern characteristic of internucleosomal DNA degradation associated with apoptosis, whereas the cellular DNA of the cells showing a high level of fluorescence (HO‐High) showed a narrow high molecular weight band. Multiparameter analysis further indicated that cells with HO‐High characteristics possessed corresponding high‐FSC/low‐SSC properties, whereas HO‐Low cells formed a cluster of low‐FSC/high‐SSC cells. Analysis of the DNA extracted from cells sorted on the basis of scatter properties alone confirmed that the low‐FSC/high‐SSC population contained the apoptotic cells and that the high‐FSC/low‐SSC population was comprised of viable cells. This methodology allowed us to determine the percentage of apoptotic cells following anti‐CD3 activation at various time points and to discriminate them from those in cell cycle. We could further quantitate the number of apoptotic versus viable CD4+ and CD8+ cells in the cell cycle. © 1993 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.

Từ khóa


Tài liệu tham khảo

10.1146/annurev.iy.10.040192.001411

10.1002/cyto.990130802

10.1016/0167-4889(92)90048-G

Duke RC, 1991, Morphological and biochemical assays of apoptosis

10.1016/0092-8674(89)90503-5

10.1016/0008-8749(92)90238-K

10.1126/science.2125368

Kim YH, 1992, Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes, J Immunol, 149, 17, 10.4049/jimmunol.149.1.17

10.1038/353858a0

10.1002/cyto.990130803

10.1038/343642a0

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

10.1038/347286a0

Odaka CH, 1990, T cell receptor‐mediated DNA fragmentation and cell death in T cell hybridomas, J Immunol, 144, 2096, 10.4049/jimmunol.144.6.2096

10.1002/eji.1830210214

Shapiro HM, 1988, Practical Flow Cytometry

10.1038/337181a0

10.1016/0022-1759(91)90396-W

10.1002/cyto.990130205

Ucker DS, 1989, Activation‐driven T cell death. I. Requirements for de novo transcription and translation and association with genome fragmentation, J Immunol, 143, 3461, 10.4049/jimmunol.143.11.3461

Ucker DS, 1992, Activation‐driven T cell death. II. Quantitative differences alone distinguish stimuli triggering non‐transformed T cell proliferation or death, J Immunol, 149, 1583, 10.4049/jimmunol.149.5.1583

Thông tin
Thông tin xuất bản

Wiley

Tập 14 Số 8

883-890

Thông tin tác giả