Treated cells in 96-well plates were incubated with BrdU labelling
solution (BrdU ELISA Kit from Roche Diagnostics, Germany), at
37 C for 2 h. The cells were fixed for 30 min, blocked with 2% BSA
for 10 min and incubated with peroxidase-labelled anti-BrdU solution
(100 ll/well) for 90 min. The colour reaction was developed
by adding tetramethylbenzidine substrate (100 ll/well) for
10 min. Absorption was read at 370 nm by ELISA reader with a reference
wavelength at 492 nm. All assays were repeated three
times.