Examination of macrophage cell surface antigen regulation by rIFN-γ and IFN-α/β utilizing digital imaging by a novel laser detection system: Anchored cell analysis station (ACAS) 470

Abstract

The use of a computer-controlled scanning laser instrument, the ACAS 470, for the analysis of cell-surface antigens on adherent macrophages is described. The association of specific cell surface antigens with macrophage differentiation and the modulation of these markers by environmental signals has long been recognized. However, direct analysis of individual macrophages for cell-surface antigen expression by conventional methods has been fraught with difficulties. Primary macrophage cultures form highly adherent monolayers in vitro. To be analyzed by conventional flow cytometric methods (e.g., FACS analysis of non-adherent lymphoid populations), the adherent macrophages must be detached by enzymatic or mechanical methods which can result in damage to the cell membranes and/or strip off cell surface antigens. Other conventional techniques, e.g., antibody plus complement-mediated cytotoxicity or immunofluorescent microscopy, are semiquantitative at best. Surface antigen analysis using ELISA techniques provides a total population measure of changes in cell surface antigen expression, but fails to provide the number of antigen-positive cells or the density of antigen per cell. The ACAS 470 obviates all of these technical problems and allows for an analysis of individual adherent cells for the density and topography of antigen per cell, as well as expression of an antigen within a population. Using the ACAS 470, we have examined the expression of Ia antigens (class II major histocompatibility antigens) and the Mac-1 antigen (C3bi receptor) on thioglycollate-elicited murine macrophages, basally and after treatment with interferons. In this study, the density and distribution of these antigens per cell, as well as their expression within a population, are reported.