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The measurement of intracellular antigens and DNA by multiparametric flow cytometry
Author(s) -
CAMPLEJOHN R. S.
Publication year - 1994
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1994.tb03493.x
Subject(s) - antigen , intracellular , proliferating cell nuclear antigen , flow cytometry , staining , paraformaldehyde , biology , cell , microbiology and biotechnology , dna , chemistry , immunology , biochemistry , genetics , organic chemistry
Summary The aim of this paper is to provide a strategy for measuring intracellular antigens combined with DNA content in cells or nuclei. A series of protocols are included which enable the majority of such antigens to be labelled and further information is provided for cases in which the standard methods prove to be inadequate. The basic principles of cell permeabilization/fixation are described, thus explaining how methods can be divided into three basic categories: (a) alcohol fixation with or without detergent pretreatment; (b) paraformaldehyde fixation followed by permeabilization with alcohol or detergents; (c) permeabilization of unfixed cells. The preparation of nuclear suspensions from paraffinembedded material is described and the possibilities and problems of staining such suspensions for nuclear antigens are discussed. Examples of results obtained with the detailed protocols are given for staining with antibodies directed against proliferating cell nuclear antigen (PCNA), Ki‐67 antigen and Ki‐Sl antigen. Details of published studies of a variety of intracellular antigens are given in two tables. The power of multiparametric flow cytometry in the study of cell proliferation, differentiation and response of cells to damage is underlined.