Summary
A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.
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References
Alavaikko M (1971) Selective separation of mouse epidermal cells and cytophotometric evaluation of their nuclear DNA. Acta Pathol Microbiol Scand A 226:9–76
Brysk MM, Snider JM, Smith EB (1981) Separation of newborn rat epidermal cells on discontinuous isokinetic gradients of PERCOLL. J Invest Dermatol 77:205–209
Cowden RR (1957) Methods for the study of the cytochemistry of nucleic acids and chromosomes. Mikroskopie 12:11–21
Filkuka J, Lederer B, Sandritter W (1968) Loss of basic nuclear protein during fermentative isolation of epidermal cells. Acta Histochem 29:263–269
Fischer SM, Nelson KDG, Reiners Jr JJ, Viaje A, Pelling JG, Slaga TJ (1982) Separation of epidermal cells by density centrifugation: a new technique for studies on normal and pathological differentiation. J Cutan Pathol 9:43–49
Fujita S (1973) DNA cytofluorometry on large and small cell nuclei stained with pararosaniline Feulgen. Histochemie 36:193–199
Fukuda M, Nakanishi K, Sawamura F, Fujita S (1977) Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry. Histochemistry 52:119–127
Giovanella BC, Heidelberger C (1965) Mouse epidermal cells and carcinogenesis. I. Isolation of skin constituents. Cancer Res 25:161–181
Goldenhersch MA, Good RA, Sarkar NH, Safai B (1982) Separation of epidermal cells by density gradient centrifugation on a continuous colloidal silica (Percoll) gradient. Anal Biochem 119:246–252
Gumucio J, Feldkamp C, Bernstein IA (1967) Studies on localization of “histidine-rich” peptide material present in epidermis of the newborn rat. J Invest Dermatol 49:545–551
Haag D, Tschahargane C, Ehemann V (1975) Isolation of single cell nuclei from human epidermis for cytophotometric DNA—measurements. Arch Dermatol Res 253:301–310
Laerum OD (1969) Oxygen consumption of basal and differentiating cells from hairless mouse epidermis. J Invest Dermatol 52:204–211
Moore JI, Karasek MA (1971) Isolation and properties of a germinative and a non-germinative cell pupulation from postembryonic mouse, rabbit, and human epidermis. J Invest Dermatol 56:318–324
Pertoft H, Laurent TC (1977) Isopycnic separation of cells and cell organelles by centrifugation in modified colloidal silica gel gradients. In: N Catsimpoolas (ed) Methods of cell separation, vol 1, Plenum Press, New York, pp 25–65
Rowden G (1975) Ultrastructural studies of keratinized epithelia of the mouse. III. Determination of the volumes of nuclei and cytoplasm of cells in murine epidermis. J Invest Dermatol 64:1–3
Sasai Y, Kumano S, Namba K, Agatsuma Y (1978) Selective separation of epidermal cells for cytofluorometric estimation of nuclear DNA. Acta Histochem Cytochem 11:418–426
Sasai Y, Kawamura K, Namba K (1979a) Loss of nuclear acid during fermentative treatment of human lymphocytes. Kurume Med J 26:11–14
Sasai Y, Kawamura K, Namba K (1979b) The separation of basal and differentiating cells from human epidermis for DNA cytofluorometry. Histochemistry 63:265–272
Stern IB, Sekeri-Pataryas KH (1972) The uptake of14C-leucine and14C-histidine by cell suspensions of neonatal rat epidermis. J Invest Dermatol 59:251–259
Sun T-T, Green H (1976) Differentiation of the epidermal keratinocyte in culture: fermentation of the cornified envelope. Cell 9:511–521
Tsanev RG, Markov GG (1960) Nucleic acid content in mouse epidermis separated from dermis by different methods. Experientia 16:448
Vaughan FL, Bernstein IA (1971) Studies of proliferative capabilities in isolated epidermal basal and differentiated cells. J Invest Dermatol 56:454–466
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Sasai, Y., Hachisuka, H., Mori, O. et al. Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry. Histochemistry 80, 133–136 (1984). https://doi.org/10.1007/BF00679986
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DOI: https://doi.org/10.1007/BF00679986