Figure 4. SA-β Gal staining in RTEC treated with C5a with or without C5aR1 blocking. SA-β-Gal activity in early passage RTEC exposed to 100 nM C5a for 3h (B, F) or 24h (J). For C5aR1 inhibition, mouse monoclonal anti-C5aR1 was pre-incubated for 1h before the C5a exposure and then maintained in fresh medium for 24h or 48h (C, G, K). More SA-β-gal+ cells were observed after C5a exposure, senescent RTEC appeared enlarged and morphologically distinct from the normal cells at the same passage with formation of larger and polynucleated cells (B and F, arrows). Untreated cells are also named as Basal (A, E, I). H2O2 exposed cells were used as positive control of senescence (D, H, L). Representative images were acquired by phase contrast microscopy. (M) Quantification of SA-β-Gal+ cells cultures. The ratio of cells positive for SA-β-gal activity was calculated by examining five not overlapping fields per condition (6-well plate). The results are presented as the mean ± SD of three independent experiments (*p< 0.05), Magnification 40X.