Rat igg2b isotype control

Fluorescence-activated cell sorting (FACS) analysis quickly evaluated microglial activation by detecting CD11b expression. Briefly, N9 cells were washed three times in a flow buffer (PBS containing 0.1% (w/v) sodium azide and 1% (w/v) BSA). The cells were then incubated with goat serum (Zhongshan Goldenbridge Biotechnology (ZsBio), Beijing, China) for 20 min at 4°C to block the non-specific binding to Fc receptors. Cells were then spun (5,000 rpm, 5 min) and washed three times in the flow buffer. Subsequently, the cells were incubated with the rat anti-mouse monoclonal antibody to CD11b (1:100) (AbD Serotec, Oxford, UK) or the rat IgG2b isotype control (1:100) (AbD Serotec) for 1 h at 4°C. Goat anti-rat IgG-DyLight®549 (1:200) (AbD Serotec) was used as a secondary antibody. Cells were resuspended in ice-cold flow buffer (250 μl). Quantitative analysis was performed using a FACSCalibur system (BD Biosciences, San Jose, CA, USA).

Fluorescence-activated cell sorting (FACS) analysis was used to determine the expression of microglial marker CD11b. After RF exposure and sham exposure, microglial cells were harvested in 1.5 mL centrifuge tubes. The cells were washed three times with staining buffer (phosphate buffered saline (PBS) containing 0.1% sodium azide and 1% bovine serum albumin (BSA)) and incubated with goat serum (Zhongshan Goldenbridge Biotechnology, Beijing, China) for 20 min at 4°C. Then, the cells were incubated with rat anti-mouse monoclonal antibody CD11b (1∶100; AbD Serotec, Oxford, UK) or rat IgG2b isotype control (1∶100; AbD Serotec) for 30 min at 4°C. Following three washes with staining buffer, the cells were then incubated with fluorescently labeled rabbit anti-rat IgG (1∶100; Invitrogen, Carlsbad, CA, USA) for 30 min at 4°C in the dark. After the final wash, cells were fixed with 2% paraformaldehyde and analyzed using a flow cytometer (BD Biosciences, San Jose, CA). Three independent experiments were performed.