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J Immunol Methods. 1997 Dec 1;209(2):111-23.
Strategies for phenotyping apoptotic peripheral human
lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric
staining methods.
Lecoeur H, Ledru E, Prévost MC, Gougeon ML.
Département SIDA et Rétrovirus, Institut Pasteur, Paris,
France.
The present article compares the reliability of four
previously described cytofluorometric methods of apoptosis
quantification for phenotyping apoptotic human lymphocytes. Each
of these assays detects distinct cellular alterations of the
apoptotic process. Alteration in plasma membrane integrity can
be evaluated following 7-AAD incorporation and the translocation
of phosphatidylserine from the inner to the outer layer of the
plasma membrane can be detected through the FITC annexin V
staining. DNA strand breaks in apoptotic nuclei can be evidenced
by the ISNT assay and finally morphological modifications can be
followed with FSC/SSC criteria. Comparative analysis of
apoptosis in cultured PBMC from HIV-infected patients
considering the FSC/SSC parameters, 7-AAD stainability and
annexin V fixation revealed that the latter identifies early
apoptotic cells, also characterized as 7-AAD(low) with a reduced
FSC. Moreover these three methods proved to be reliable and gave
statistically similar results when combined with cell surface
detection of antigens such as CD4, CD8 and CD19 by specific
mAbs. Importantly, the 7-AAD assay easily allowed the
identification of debris/apoptotic bodies, which were still
stained by anti-cell surface mAbs and might therefore
significantly distort the apoptosis percentage in a given
lymphocyte subset. In the present report we also point out that
the ISNT assay is not appropriate for phenotyping apoptotic
lymphocytes in PBMC. Indeed it can particularly underestimate
the rate of apoptosis in the B-cell subset. This was found to be
related to the apoptosis-associated decrease in cell surface
antigen expression, which is dramatically exacerbated in the
ISNT assay because of the stripper effect of ethanol used for
cell permeabilization. Finally, we propose a three step
analytical strategy to accurately phenotype apoptotic peripheral
human lymphocytes. It includes two gating steps performed on
FSC/SSC criteria and 7-AAD/FSC parameters to eliminate
monocytes, granulocytes and debris-apoptotic bodies, the third
step being the phenotyping step itself, performed in dual or
triple staining experiments. Altogether these observations
emphasize that it is essential to assess critically the ability
of a cytofluorometric method to phenotype apoptotic cells in
complex lymphoid populations and that inaccurate identification
of cell subsets undergoing apoptosis can be readily overcome by
gating properly the lymphoid population, and using assays which
preserve cell surface structure.
PMID: 9461328 |
