Figure 5
LLLT-mediated microglial phagocytosis is a Rac-1-dependent actin-based process. (A) LPS-activated BV-2 cells pretreated with or without wortmannin (100 nM) under LLLT treatment. 30 min after incubation, cells were fixed with 4% paraformaldehyde and stained with FITC-labeled phalloidin to visualize F-actin (green), and confocal Z-stacks were acquired. Images shown are representative of approximately 100 cells. Bar = 50 μm. (B) Primary microglia transfected with Rac1Q61L, Rac1T17N or wt-Rac1. G418-resistant cells were collected for further LPS stimulation. 30 min after LLLT (20 J/cm2) treatment, total F-actin was stained using FITC-phalloidin and measured using a 96-well plate reader. F-actin ratio was calculated as (F-actin in transfected cells - background)/(F-actin in wt-Rac1 transfected cells - background). Data represent mean ± SEM (n = 6). (C) Representative western blot analysis of LLLT-treated LPS-activated BV-2 cells to detect GTP-Rac1 and total Rac1. (D) Quantitative analysis of the levels of GTP-Rac1 and total Rac1 in treated cells. Data represent mean ± SEM (n = 4; *P <0.05 versus control cells). LLLT, low-level laser therapy; LPS, lipopolysaccharide.