MULTIPARAMETER FLOW CYTOMETRIC CHARACTERIZATION OF EPIDERMAL-CELL SUSPENSIONS PREPARED FROM NORMAL AND HYPERPROLIFERATIVE HUMAN SKIN USING AN OPTIMIZED THERMOLYSIN TRYPSIN PROTOCOL

Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions, Several methods to separate t he dermis from the epidermis are available, The proteolytic enzyme the rmolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation, In the prese nt study an optimized cell isolation procedure using thermolysin and t rypsin is described, which is particularly suitable for punch biopsies , A 16-20-h (overnight) incubation of biopsies taken from normal and h yperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C pro duced a selective separation of the dermis and epidermis, After a 30-m in trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension w as produced which was characterized by minimal cell damage (cellular d ebris and clumps), a high recovery of basal cells and high quality DNA histograms, Furthermore, dermal contamination was very low, The therm olysin-trypsin separation methodology followed by triple-labelling flo w cytometry provided a precise quantification of the percentage of ker atin 10-positive cells, vimentin-positive cells and cells in S and G(2 )M phases, Proliferative activity was selectively measured in the basa l, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping , In contrast to the non-keratinocytes, the percentage of cells in S a nd G(2)M phases in the basal keratinocytes and in the suprabasal compa rtment increased 44-48 h after stripping, The increased proliferation following tape stripping was paralleled by an increased invasion of vi mentin-positive cells into the epidermis and preceded by a decreased n umber of keratin 10-positive cells, Thermolysin-trypsin separation fol lowed by three-colour flow cytometry permits a highly selective charac terization of normal and hyperproliferative epidermis.