Flow cytometry for separation of keratinocyte subpopulations from the viable epidermis

Abstract

Human epidermal cell suspensions were analyzed and sorted with flow cytometry. The desmosome and differentiation-related KM48 monoclonal antibody was used for indirect immunofluorescence and permitted staining of keratinocytes at various stages of the cell maturation. Intensity of the staining correlated with the degree of differentiation. Three sorting gates were chosen to obtain subpopulations which varied distinctly in KM48 expression. The flow cytometry-sorted cells were characterized by their ultrastructural appearance and by the bullous pemphigoid antigen expression. According to the ultrastructure criteria, about 50% of the cells obtained from the "IF negative" gate were basal layer keratinocytes (45.5% expressed bullous pemphigoid antigen); 90% of the "intermediate" gate cells were spinal layer keratinocytes, and over 80% of the cells sorted through the "strongly IF positive" gate were of the granular layer type. The method of keratinocyte separation proposed allows samples to be obtained for further biochemical and functional studies on keratinocyte subpopulations in normal and pathologic skin.