Improved permeabilization procedure for flow cytometric detection of internal antigens. Analysis of interleukin-2 production.

Abstract:

:A cell membrane permeabilizing treatment is described which involves the use of lysolecithin at low concentration in acidic acetate buffer and paraformaldehyde fixation. It preserved well-separated scatter cytograms of small and large lymphocytes. The accuracy of the immunochemical detection of internal antigens by flow cytofluorography was demonstrated by the linear relationship between the percentage of fluorescent cells detected and the proportion of intracellular antigen-containing cells in mixtures with antigen-negative cell lines. Cell cycle analysis by dual nuclear staining with propidium iodide and FITC-conjugated Ki-67 antibody recognising in vitro stimulated human T lymphocytes verified that the proliferating lymphocytes retained their increased light scatter properties after permeabilization. Enumeration of interleukin-2 (IL-2) producing cells by their cytoplasmic immunofluorescence showed that enlarged lymphocytes were the main IL-2 producing cells. This improved permeabilization procedure, by gating small and enlarged lymphocytes separately, makes it possible to determine by two color fluorescence the immunophenotype of activated T cells committed to interleukin production.

journal_name

J Immunol Methods

authors

Labalette-Houache M,Torpier G,Capron A,Dessaint JP

doi

10.1016/0022-1759(91)90162-9

subject

Has Abstract

pub_date

1991-04-25 00:00:00

pages

143-53

issue

2

eissn

0022-1759

issn

1872-7905

pii

0022-1759(91)90162-9

journal_volume

138

pub_type

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