A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation.
This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.
van Erp
P E
PE
Department of Dermatology, University Hospital, Nijmegen, The Netherlands.
Brons
P P
PP
Boezeman
J B
JB
de Jongh
G J
GJ
Bauer
F W
FW
D001853:Bone Marrow / Q000032:analysis / Q000187:drug effects; D001854:Bone Marrow Cells; D001973:Bromodeoxyuridine; D004247:DNA / Q000032:analysis*; D000078404:Epidermal Cells; D004817:Epidermis / Q000032:analysis / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D007633:Keratins; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism* / Q000494:pharmacology; D011489:Protein Denaturation
D007633:Keratins; D004247:DNA; D010447:Peptide Hydrolases; D001973:Bromodeoxyuridine