An easy method for preparation of bare cell nuclei from fresh solid tissues for
DNA flow cytometry is described. Pieces of up to 2 x 2 x 2 mm3 size from fresh tissues were fixed in
formalin. After removal of
formalin by washing with
ethanol and
rehydration with tap water, the tissue pieces were incubated with
subtilisin Carlsberg (
pronase, Sigma
protease XXIV) and then stained directly with
DAPI. Staining with
ethidium bromide gave unsatisfactory results. Neither mechanical disaggregation nor centrifugation were used. The resulting cell nucleus
suspensions had extremely low frequencies of debris particles and of clumped cell nuclei. A good yield, a minimized loss, and a good stainability of cell nuclei were obtained. The applicability of the method was exemplified by the analysis of biopsies from the colon-rectum in patients with
ulcerative colitis and of biopsies from the bladder in patients with
bladder cancer and compared to the standard method of this laboratory, which uses mechanical disaggregation,
ethanol fixation,
pepsin treatment, and staining with
ethidium bromide. The
formalin-
subtilisin Carlsberg technique resulted in good agreement of ploidy measurements compared to the standard method, a higher number of evaluable histograms, an improved detectability of
aneuploid cell populations, and an improved accuracy of the S- and G2-phase analysis, particularly in samples with low proliferation. The method also makes it possible to use long-term storage and to transport samples by post.