THE USE OF ESCHERICHIA-COLI EXONUCLEASE-III TO GENERATE SINGLE-STRANDED-DNA IN BRDURD CELL-CYCLE ANALYSIS PERMITS SIMULTANEOUS DETECTION OF CELL-SURFACE ANTIGENS

An immunocytochemical method for the simultaneous flow cytometric quantitation of total cellular DNA, incorporated 5-bromo-2′-deoxyuridine (BrdUrd) and one or more cell surface antigens has been developed. Biotin labeling of cell surface antigens, critically tuned fixation techniques and an enzymatic denaturation of cellular DNA are the essential features of this method. Enzymatic deneturation of cellular DNA was shown to prevent loss of cell surface antigen-bound biotin moieties, and thus to preserve cell surface immunofluorescence distribution. After a mild protein extraction and the introduction of breaks into the chromatin using restriction endonucleases, E. coli exonuclease III was used to generate stretches of single stranded DNA. This approach permits detection of the incorporated BrdUrd using anti-BrdUrd monoclonal antibodies. The enzymatic denuturation protocol was optimized using in vitro BrdUrd-labeled L1210 murine leukemia cells, and applied to both in vivo and ex vivo BrdUrd-labeled murine bone narrow cells. With this new method it id possible to study DNA content, cell cycle kinetics and cell surface antigen expression simultaneously, and hence functional relationships between these parameters can be investigated. © 1990.