Detection of intracytoplasmic antigens by flow cytometry

A new technique is described for the detection of intracellular antigens by immunofluorescence and flow cytometry. The technique utilizes lysolecithin (lysophosphatidylcholine), a naturally occurring phospholipid, to permeabilize cell membranes and allow antibodies to reach intracellular antigens. The technique is rapid and sensitive, and retains sufficient integrity of the cells being treated to enable differentiation of cell types on the basis of light scatter (e.g., lymphocytes from monocytes). Permeabilization of cells following lysolecithin was assessed using standard techniques including trypan blue exclusion, propidium iodide staining, and hydrolysis of fluorescein diacetate. Lysolecithin treatment was accompanied by only minimal increases in non-specific background fluorescence, and no increase in autofluorescence. Our studies have demonstrated that lysolecithin treatment of human mononuclear cell populations permits flow cytometric analysis of cytoskeletal structures, including intermediate filaments, as well as cytoplasmic immunoglobulin. Studies currently in progress in our laboratory have demonstrated broad intracellular reactivity with some monoclonal antibodies identifying leukocyte differentiation and tumor-associated antigens. These findings contrast with the more restricted expression of these antigens on the cell surface and demonstrate not only the value of the lysolecithin technique but also the importance of the study of intracellular antigens in our overall understanding of the specificity, distribution, synthesis, and function of cellular antigens.