Simultaneous Cytometric Analysis For The Expression Of Cytoplasmic And Surface-antigens In Activated T-cells

Abstract

A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates. Fluorochrome-antibody conjugates bound to cell-surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA. STA is a unique determinant detected in activated human T cells by the monoclonal antibody K-1-21, which also recognizes a cross-reactive conformation-dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxic/suppressor T cells and Leu 3a+ helper/inducer T cells but is not induced in the Leu 15+ population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells.