Anti brdu monoclonal antibody

DNA synthesis in proliferating cells was determined by measuring BrdU incorporation with a BrdU ELISA assay [28 (link)]. For this purpose, the cells were seeded in 96-well culture plates at a density of 10,000 cells/well. Phosphatidylcholine nanoparticles at 0.1 and 0.01% were added in 100 μL of medium in absence of serum. Cells were further incubated at 37°C for 48 hours in a 5% CO₂ atmosphere. BrdU (0.01 M final concentration) was added to the cells 16 hs before the end of incubation with PC nanoparticles. At the conclusion of labeling, cultures were rinsed with phosphate buffered saline (PBS), pH 7.0, fixed with 70% EtOH, denatured with 2 M HCl (100 μL/well, 10 minutes, 37°C), and neutralized with 0.1 M Trizma buffer, pH 9. Cells were then incubated with monoclonal anti-BrdU antibody (50 μL/well; 1 μg/mL final; Roche, USA) at 37°C for 60 minutes, washed with PBS, and incubated with goat anti-mouse IgG horseradish peroxidase (HRP) conjugate at 37°C for 30 minutes. Afterwards, cells were washed and labeling evidenced with tetramethylbenzidine (TMB). Triplicates were run for each treatment. Values were expressed in terms of percent of untreated control cells.

BrdU incorporation assays were conducted as described previously [56 (link), 58 (link)]. Briefly, cells were labeled with 10 μM BrdU for 10 hours and then fixed with 95% ethanol and 5% acetic acid and treated with 2M HCl containing 1% Triton X-100. The cells were stained with monoclonal anti-BrdU antibody (Roche), followed by staining with Alexa Fluor 546 (red) goat anti-mouse antibodies and 4′, 6′-diamidino-2-phenylindole (DAPI). Stained cells were imaged in five randomly selected fields with an EVOS fluorescence microscopy. The BrdU-positive cells were counted and quantified using the ImageJ software.