Rapid detection of S-phase cells by anti-bromodeoxyuridine monoclonal antibody in 9L brain tumor cells in vitro and in situ

Abstract

FITC-conjugated anti-bromodeoxyuridine (BrdUrd) monoclonal antibody (anti-BU-MAb) was used to detect S-phase cells of 9L rat brain tumor cells in vitro and in situ. Monolayer 9L cells were treated with 0.625–20 μM of BrdUrd for 30 min, harvested, and reacted with a 1:100 dilution of FITC-conjugated anti-BU-MAb and analyzed with a flow cytometer. BrdUrd-treated cells stained satisfactorily with antibody. Values obtained for the labeling index using this method (48.6%) were 10%–20% higher than the fraction of cells in S-phase calculated from DNA histograms or as the labeling index calculated from autoradiographs of cells pulse-labeled with³H-thymidine. BrdUrd (1–40 mg/kg) was administered by i.p. injection to rats bearing 9L brain tumors. Single cell suspensions obtained by disaggregation of excised tumors were stained with anti-BU-MAb. The percent-age of fluorescent cells (15.9%) calculated using this method was similar to that of S-phase cells (17.2%) calculated from DNA histograms and from autoradiographics for tumor bearing rats pulse-labeled with³H-thymidine in situ. The antibody staining technique is a rapid and accurate method for various cell kinetic studies both in vitro and in vivo in a rat model, and has promise as a technique for the study of cell kinetics in humans.