title: Preparation of cell nuclei from fresh tissues for high-quality DNA flow cytometry.
abstract: 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.
authors.affiliation: Department of Medical Radiobiology, Karolinska Institute, Stockholm, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Castro
authors.forename: T
authors.initials: T
authors.lastname: Heiden
authors.forename: N
authors.initials: N
authors.lastname: Wang
authors.forename: B
authors.initials: B
authors.lastname: Tribukait
mesh_terms: D000818:Animals; D001706:Biopsy; D002453:Cell Cycle; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000737:chemistry; D003093:Colitis, Ulcerative / Q000473:pathology; D003106:Colon / Q000648:ultrastructure; D004247:DNA / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis; D000431:Ethanol; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005557:Formaldehyde; D006801:Humans; D007211:Indoles; D051379:Mice; D010434:Pepsin A; D011003:Ploidies; D013381:Subtilisins; D001743:Urinary Bladder / Q000648:ultrastructure; D001749:Urinary Bladder Neoplasms / Q000473:pathology
chemical_list: D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D007211:Indoles; D005557:Formaldehyde; D000431:Ethanol; C007293:DAPI; D004247:DNA; D013381:Subtilisins; D010434:Pepsin A