BrdU incorporation assay

DNA synthesis in proliferating cells was determined by measuring 5-Bromo-2-deoxyUridine (BrdU) incorporation. BrdU assays were performed at 24 h and 48 h after infection. After seeding the infected cells in 96-well culture plates at a density of 2 × 10³ cells/well, they were cultured for 24 h or 48 h, and incubated with a final concentration of 10 μM BrdU (BD Pharmingen, San Diego, CA, USA) for 2 h to 24 h. When the incubation period ended, we removed the medium, fixed the cells for 30 min at RT, incubated them with peroxidase-coupled anti-BrdU-antibody (Sigma-Aldrich) for 60 min at RT, washed them three times with PBS, incubated the cells with peroxidase substrate (tetramethylbenzidine) for 30 min, and measured the absorbance values at 450 nm. Background BrdU immunofluorescence was determined in cells not exposed to BrdU but stained with the BrdU antibody.