Separation of mouse epidermal basal and differentiating cells for microflow fluorometric measurements

Abstract

The DNA content of lymphocytes and of basal cells from normal hairless mouse epidermis was measured by microflow fluorometry (MFF). To obtain a relatively pure suspension of epidermal basal cells a combined mechanical and enzymatic method was used. The admixture of differentiating cells into the basal cell fraction after cell separation was 13%. The results were compared with those obtained with conventional Feulgen microspectrophotometry applied to basal cells and dermal lymphocytes in histologic sections. The results from both cytophotometric methods were in good agreement and clearly demonstrated the improved resolution obtained by using MFF. When the lymphocytes were not treated with pepsin before being stained with ethidium bromide for MFF, the modal DNA value was consistently below that of the basal cells from the same specimen. Pepsin treatment of lymphocytes, however, incresed their fluorescence intensity to the value of epidermal basal cells. The modal DNA value of Feulgen-stained dermal lymphocytes in histologic sections was consistently below that of epidermal basal cells from the same section. The advantage of pepsin treatment for obtaining higher resolution of DNA measurements of basal and differentiating epidermal cells and of lymphocytes was evaluated. The cell cycle distribution of basal cells from epidermis in different states of proliferative activity was determined. Changes in the proportion of cells in S phase were parallel to changes in the 3H-Tdr [tritiated thymidine] labeling index.