Modification of a trypsin‐detergent method for DNA flow cytometry of human epidermis

Abstract

An existing technique (Vindelov et al. Cytometry 3:323, 1983) has been modified for DNA flow cytometry of human epidermis obtained from 2 to 3 mm punch biopsies. By varying the length of time of digestion of the epidermal disc by trypsin from 5 to 70 Key terms: Trypsin digestion, biopsy, keratimin a controlled release of keratinocytes occurred beginning with the stratum basale and proceeding toward, but not including, the superficial layer of the epidermis, the stratum corneum. nocyte, isolated nucleiAn important prerequisite for performing flow cytometry (FCM) is the acquisition of a single cell or nuclear suspension. For epidermis, the production of single cell suspensions permits the study of a variety of cell types such as Langerhans cells, keratinocytes, keratinocyte subpopulations, and basal cells (2-11). Because of our interest in the possibility of using cell cycle kinetic data obtained from human epidermis as indicators of the best circadian time for maximal host tolerance to chemotherapy, i.e., chronochemotherapy (11, we modified a n existing technique (12) for use in DNA FCM of human epidermis.Dispersion of epidermal cells or their nuclei into suspension has been accomplished by several different methods including trypsin, dithiotheritol (DTT), EDTA, ultrasonication, or ultrasonication plus DTT (2). The trypsin detergent method described by Vindelov et al. (12) has the advantages of (a) frozen storage of the specimen, (b) production of clean nuclei after digestion of cytoplasmic proteins with trypsin, and (c) quantitative staining of DNA with propidium iodide.This report describes a modification of an existing technique (12) for the production of isolated nuclei of keratinocytes obtained from 2-3 mm punch biopsies of adult human epidermis. Based on the duration of the incubation time in trypsin, a controlled release of keratinocyte populations can be achieved. Trypsin digestion dissolved epidermal cell cohesion beginning in the basal cell layer with progression toward the superficial-most layer after longer incubation. MATERIALS AND METHODS BiopsiesThe skin biopsies used in this study were obtained from the scalp during hair transplantation surgery. Epidermal-Dermal Separation and Freezing of Isolated EpidermisTo facilitate the removal of epidermis from dermis, skin biopsies were first individually placed in 0.5% cold acetic acid for 48 h. With the aid of a dissecting microscope, the epidermis was separated from the dermis using watchmaker's forceps. The disc of epidermis was then placed in fresh DMSO solution and frozen rapidly to -70" C. Intact biopsies as well as the peeled dermis and epidermis were stained with hematoxylin and eosin (H & E) and examined by routine light microscopy. Trypsinization and StainingThe trypsin-detergent method of Vindelov (12) was used with the modification that digestion times in trypsin were varied from 5 to 70 min. In addition to varying the digestion time, other variables which were studied included mechanical vortexing at 1 min, 3 min and 5 min intervals during th...

Flow cytometric DNA analysis of corneal epithelium

“…The S fit cell-cycle program analysis of the rabbit DNA histogram calculated the following values: digestion of the sheet of epithelium. Increased exposure of the tissue to the trypsin has been used by us to successfully prepare human epidermis for FCM (Stough et al, 1989). In the second step, trypsin inhibitor and ribonuclease A were added for 10 additional min.…”