Quantitative analysis of HLA-DR antigen expression by normal human epidermal cells (light microscopy and flow cytometry sorting)

Abstract

This chapter focuses on quantitative analysis of human leukocyte antigen-DR antigen expression by normal human epidermal cells. Crude epidermal cell (EC) suspensions were prepared from freshly removed normal skin. Small slices of tissue were chopped and incubated in 0.3% trypsin for 1 h at 37°C. The epidermis was then detached from the dermis with fine forceps and agitated in MEM supplemented with 20% fetal calf serum. Cell aggregates and debris were removed from the single-cell suspension by filtration through a double layer of gauze. The cells were counted, and viability was assessed by trypan blue exclusion (85% or better). A portion of the cells was resuspended in RPMI 1640 supplemented with 15% heat-inactivated AB human serum, 20 mM Hepes buffer, 2 mM l-glutamine, and antibiotics. Dispersed crude human ECs labeled with either BL2-MCA or MAS 054-MCA revealed specific labeling of 2.0 ± 0.6% of the total normal EC. Skin cells incubated with normal mouse serum or rat serum plus the appropriate fluoresceinated immunoglobulin showed no fluorescence.