MULTIPARAMETRIC CELL-CYCLE ANALYSIS OF PERIPHERAL BLOOD-ACTIVATED LYMPHOCYTE SUBSETS USING STAINING BASED ON THE TDT METHOD FOR INCORPORATED BRDURD

This study describes a new method for the simultaneous assessment of t he distribution of a cell population in the G0/G1, S, and G2/M cell-cy cle phases by using multiparameter how cytometry and single staining b ased on BrdUrd incorporation. Both the K562 cell line and PHA-stimulat ed peripheral blood lymphocytes (PBL) were analyzed. Cells were cultur ed in the presence of BrdUrd for 30 min prior to cell harvesting. Once collected, cells were exposed to ultraviolet light for 5 min and then fixed immediately in 70% ethanol (-20 degrees C) for at least 30 min. Once fixed, the cells were placed for 30 min at 37 degrees C in the p resence of terminal deoxynucleotidyl transferase (TdT) and dUTP labele d with digoxigenin; they were then stained with FITC-labeled anti-digo xigenin. Our results show that G0/G1, S, and G2/M cell populations can be clearly discriminated according to FITC fluorescence and light-sca tter parameters. In this way, S-phase cells can be identified by their FITC staining. From the cells which were negative for anti-digoxigeni n-FITC antibody, two clear populations could be resolved in a forward scatter, side scatter, and fluorescence pulse-width three-dimensional plot; the values obtained for G0/G1 cells were lower than those obtain ed for G2/M cells in all three parameters. Multiparameter analysis of PBL stained for two surface antigens (CD3 and CD8) and for BrdUrd by d irect or indirect TdT method permitted cell-cycle analysis of differen t subpopulations, including CD3+/CD8+, CD3+/CD8-, CD3-/CD8+, and CD3-/ CD8-. (C) 1996 Wiley-Liss, Inc.