The measurement of intracellular antigens and DNA by multiparametric flow cytometry

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 paraffin-embedded 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-S1 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.