See Abreview

See Abreview

Cells were pulse labeled with 10 mM BrdU for 30 min, rinsed twice in prewarmed PBS, and chased in prewarmed culture medium, supplemented with 5 mM deoxythymidine. Incorporated BrdU was detected after ethanol fixation of the cells, which were than rinsed once in PBS and resuspended in 2 ml of 0.4 mg/ml pepsin in 0.1 N HCl. After 30 min at room temperature cells were pelleted, resuspended in 2 N HCl, and incubated for another 30 min at 37°C. Cells were rinsed in 0.1 M borate buffer, pH 8.5, and PBS/BSA (1 mg/ml BSA in PBS). Appropriately diluted mouse anti-BrdU antibody (clone IIB5) was added to the cell pellet, resuspended in 100 micro liters PBS/BSA. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization, FITC-conjugated Fab2 fragments of rabbit anti-mouse Ig antibody were added in a 1/10 dilution. After incubation for 45 min at room temperature samples were rinsed twice in PBS/BSA and the cells were finally resuspended in 0.5 ml cold PBS supplemented with 100 microgram/ml RNAse and 20 µg/ml propidium iodide. The samples were allowed to stand for 15 min on ice in the dark before flow cytometric analysis. In the negative control the primary antibody was omitted.