COMPARISON OF VINDELOV ET-AL AND BROMODEOXYURIDINE DNA DOUBLE-STAINING FLOW-CYTOMETRY METHODS FOR ANALYSIS OF CELL-CYCLE DISTRIBUTION IN RAT THYMOCYTES/

This study compares the cell cycle distribution in rat thymocytes obta ined by means of bromodeoxyuridine (BrdUrd) labeling of S-phase cells and the analysis of the S-phase fraction obtained according to the tec hnique of Vindelov et al. (Cytometry 3:332-338, 1983). The proportion of BrdUrd-labeled cells was analyzed in single cell suspensions of adu lt rat thymocytes after in vivo injection of BrdUrd and the results th en compared with those obtained after measuring the cell DNA contents according to the Vindelov et al. method. The percentage of BrdUrd-posi tive cells was greater than the S-phase fraction obtained using the Vi ndelov et al. technique. By contrast, no major differences were observ ed between the percentage of BrdUrd-positive cells and the S-phase fra ction obtained after analyzing the DNA histograms of the same data fil es with the RFIT mathematical model. The elimination of trypsin treatm ent used in the Vindelov et al. method did not alter the results, wher eas the use of DNA denaturation with 2N HC1 was shown to increase the percentage of S-phase mt thymocytes (calculated from DNA histograms) i ndependently of whether trypsin treatment was used or not. However, th e value of the S-phase fraction was not as gr eat as that obtained aft er BrdUrd labeling. Thus when comparing BrdUrd-labeling and the Vindel ov et al. technique, important differences in the percentage of S-phas e adult rat thymocytes were observed. Selective G0/G1 cell loss during washing and centrifugation steps performed after the DNA denaturation used for BrdUrd detection was the main reason for these differences. (C) 1996 Wiley-Liss, Inc.