Monoclonal anti brdu antibody clone bu 33

Bromodeoxyuridine (5-bromo-2′-deoxyuridine; BrdU) is known to be incorporated into the newly synthesized DNA of replicating cells during the S phase of the cell cycle. To identify actively replicating cells, and thereby assess cellular proliferation, BrdU was applied to cells at days 7, 10, and 12 of culture. To visualize BrDU, cultures were incubated for 2 h with a monoclonal anti-BrdU antibody (clone BU 33; Sigma). After fixation with methanol for 10 min, the cells were treated with 1 N HCl for 1 h at room temperature before incubation with a TRITC-conjugated secondary antibody (Sigma) for 1 h in a dark room. Finally, the cells were stained with 1 mg/mL 4′,6-diamidino-2-phenylindole (DAPI; Vectashield; Vector Laboratories, Burlingame, CA). Immunofluorescence was observed using a confocal microscope (Carl Zeiss, Jena, Germany) and images were acquired using AxioVision software (Carl Zeiss). Images (n = 3 for each experimental conditions) were converted to binary and analyzed using ImageJ software (version 1.44; National Institutes of Health, Bethesda, MD).

FUrd is based on the incorporation of a fluorine-conjugated uridine analogue and reflects that rRNA comprises more than 80% of cellular RNA. Cells were incubated with 2 mM FUrd (Sigma-Aldrich, cat. no: F5130) in DMEM and incubation was allowed for 10 min at 37°C. To stop, medium was removed and cells were washed with cold PBS. For detection of incorporated FUrd, cells were fixed with a 4% formaldehyde solution in PBS at room temperature, permeabilized with a 0.5% Triton X-100 solution in PBS and incubated with Monoclonal Anti-BrdU antibody clone BU-33 (Sigma Aldrich, cat. no: B8434). Actinomycin D was used as control drug.