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We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.

~ ~The patterns of [.3HI-leucine and [32Pl-phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the GO + GI and (& + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two-dimensional polyacrylamide gel ultra-microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of ['HI-leucine-and ['2P]-phosphate-labeled protein regions from the autoradiographs occurring primarily inThe refinement of two-dimensional polyacrylamide gel electrophoresis (PAGE) has contributed significantly to the knowledge and the elaboration of gene products during cellular development and differentiation. In disciplines such as biochemistry, cell biology, teratology, etc., a need has arisen for methods to analyze single cells (eggs), small numbers of simple populations (e.g., cell cycle compartments, blastulae), and larger but relatively small numbers of complex populations (e.g., organs, embryos). The adaptation of a two-dimensional PAGE system for the analysis of minute amounts of protein from small samples has been developed (13), and the sensitivity was shown to be far greater than conventional electrophoresis techniques. For example, several hundred protein regions have been observed from a single Drosophila egg with resolution as low as 3 pg of protein (13).The objectives of the present study were, first, to assess the potential of micro-sampling for the collection of relatively small numbers of nuclei (200,000) from winthe 6 2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the Gz + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of Gz + M and early mitosis were composed, in part, of nucleolar proteins.

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...

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