Abstract:
:Murine lymphocytes readily undergo spontaneous and glucocortocoid-induced apoptosis in vitro. It has been previously demonstrated that during apoptosis, many cell types including lymphocytes, enzymatically cleave their DNA, thus demonstrating a sub-G0 DNA peak when stained with propidium iodide and analyzed by flow cytometry. In a mixed population, it is often desirable to phenotypically identify distinct populations or subsets undergoing apoptosis, thus requiring multiparameter analysis of surface phenotype and DNA content. Paraformaldehyde fixation procedures, although common for surface evaluation, have not been extensively used in methods quantifying apoptosis. To measure apoptosis in a mixed lymphocyte population, we evaluated a gentle detergent permeabilization and paraformaldehyde fixation procedure combined with propidium iodide (PI) DNA staining, adapted from existing methods for cell cycle studies. With this method and rigorous gating techniques which we defined, we detected both apoptotic and debris fractions within the sub-G0 cell cycle region of a glucocortocoid-treated murine lymphocyte cell line. Using this cell line, WEHI 231.7, as a lymphocyte model, we developed a logical gating strategy to exclude debris from analysis. We further demonstrated that apoptosis in freshly isolated murine lymphocytes detected with paraformaldehyde fixation and PI staining was quantitatively comparable to PI staining with ethanol fixation, or nick translation labeling of DNA strand breaks (TUNEL). Finally, using fresh murine spleen cells, we demonstrated that paraformaldehyde fixation preserves surface protein staining, allowing multiparameter analysis of immunophenotype and apoptotic or cell cycle status in a mixed lymphocyte population. Thus, this method offers an inexpensive and technically simple alternative for assessing apoptosis and surface phenotype.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Douglas RS,Tarshis AD,Pletcher CH Jr,Nowell PC,Moore JSdoi
10.1016/0022-1759(95)00216-2subject
Has Abstractpub_date
1995-12-27 00:00:00pages
219-28issue
2eissn
0022-1759issn
1872-7905pii
0022175995002162journal_volume
188pub_type
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(80)90126-x
更新日期:1980-01-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1991-11-05 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1996-06-10 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2019.06.019
更新日期:2019-11-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:2015-12-01 00:00:00
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journal_title:Journal of immunological methods
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更新日期:2019-04-01 00:00:00
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pub_type: 杂志文章,评审
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更新日期:2011-01-05 00:00:00
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journal_title:Journal of immunological methods
pub_type: 信件
doi:10.1016/j.jim.2014.07.011
更新日期:2014-12-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(99)00194-5
更新日期:2000-01-13 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1987-02-26 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:2011-10-28 00:00:00
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1990-03-09 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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pub_type: 杂志文章
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