Single-cell immuno-β-galactosidase staining of heterogeneous populations. Practical application on limited cell numbers

Abstract

Summary In this paper we describe a sensitive immunocytochemical staining method, particularly useful for the study of subpopulations of cells in complex mixtures such as bone marrow cell suspensions.E.coli β-galactosidase is used as a label, which has the advantage that no endogenous activity is observed under the present experimental conditions. Direct sedimentation of cells on to poly-l-lysine-pretreated multi-well slides followed by gentle fixation prevents cell loss during preparation and subsequent incubation steps. Furthermore, analysis of only a few hundred cells per sample is possible. We examined the sensitivity of this method by comparing the percentages of positive cells in a spleen cell suspension after staining with a panel of monoclonal antibodies followed by analysis with the present immuno-β-galactosidase method or standard flow cytometry. For almost all antibodies used, the percentages of positive spleen cells obtained with the immuno-β-galactosidase method at least equalled those obtained with flow cytometry. Several fixatives, used to permanently adhere the cells to the slide's surface, were tested for the preservation of both morphological and antigenic structure. Glutaraldehyde and formol acetone proved to be the best choices in this respect. The present method combines high sensitivity with good morphology and is especially useful for immunophenotyping low cell numbers of heterogeneous populations.