Open AccessDNA FLOW CYTOMETRY OF ISOLATED KERATINIZED EPITHELIA: A METHODOLOGICAL STUDY BASED ON ULTRASONIC TISSUE DISAGGREGATION
Author(s) -
Møller Ulla,
Larsen J. K.
Publication year - 1979
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/j.1365-2184.1979.tb00126.x
Subject(s) - flow cytometry , epithelium , sonication , microbiology and biotechnology , biology , ethidium bromide , dna , pathology , chemistry , chromatography , biochemistry , medicine , genetics
Ultrasonication of keratinized, stratified, squamous epithelium, which had been separated from underlying tissue by means of acetic acid, resulted in disaggregation of all cellular layers in the epithelium, giving a suspension of single nuclei with mitoses preserved. This suspension was treated with RNAse and ethidium bromide for analysis by flow cytometry. From the resulting DNA histogram the G 1 , S and G 2 + M fractions were estimated using the computer program of Fried (1976). Treatment with dithiothreitol before sonication increased the yield of nuclei in suspension and decreased the amount of debris and clumps, thereby suppressing overestimation of small S fractions. This method of preparation prior to DNA flow cytometry was useful for the study of the hamster cheek pouch epithelium and of normal and pathological human epidermis.