Microglial Activation Assay

For the morphological analysis of microglia after the addition of increasing Poly I:C-concentrations, 20.000 microglia in 500 μl medium (≙ 40.000 cells/ml) were plated out per well. Before, glass coverslips were placed in 4-well plates (Thermo Fisher Scientific Inc.: Cat. No.: 176740) and coated with 10 μg/ml (w/v) PDL (Sigma-Aldrich; Cat. No.: P0899) for 1 h at 37°C in the incubator. After culturing the microglia for 2 days at 37°C and 6% CO₂, half of the medium was removed and replaced by fresh microglia medium or by microglia medium containing 50 or 100 μg/ml Poly I:C (Sigma Aldrich; Cat. No.: P1530). Subsequent, the 4-well plates were placed in the incubator for 3 h. The incubation time and Poly I:C concentrations used in this study were adapted from a previous publication which showed that Poly I:C activates microglia in vitro by toll-like receptor 3 (TLR-3) signaling (Town et al., 2006). Then, microglia were fixed with pre-warmed 4% (w/v) paraformaldehyde (Carl Roth GmbH & Co. KG; Cat. No.: 4235.1) for 10 min. After three washing steps with PBS (1x) the cells were stored at 4°C until immunocytochemical staining were performed.