Culture and Treatment of Primary Mouse Microglia

Primary microglia were cultured from the brains of pups (postnatal day 6) of TDP-43^A315T mutant mice. Brains were collected and placed in ice-cold PBS. Following mechanical dissociation, the brains were incubated in a 0.25% Trypsine-EDTA solution (Sigma-Aldrich) containing 250 K U/ml DNase I (Sigma-Aldrich). After centrifugation, the cell pellets were placed in T-75 cm² flasks (Sarstedt, Nümbrecht, Germany) for 10 d at 37 °C, 5% CO₂, in DMEM high-glucose media with 10% fetal bovine serum and antibiotic solution (Sigma-Aldrich). Genotyping carried out from tissues of the pups confirmed the identity of each culture. At confluence, the wild-type (WT), as well as transgenic microglia, were plated in 6-well plates at a concentration of 200,000 cells/well in serum containing media. Cells were incubated with granulocyte colony-stimulating factor 24 h later to allow adhesion. After 2 to 3 days cells were deemed suitable for experimentation.

Prior to treatment, cells were transferred to serum-free media. Microglia from WT mice or transgenic TDP-43^A315T mice were challenged with LPS or DMSO for 6 h to elicit cytokine/chemokine release. One set of WT microglia was treated with LPS for 3 h followed by further addition and incubation with ASH extract in DMSO (250 μg/ml) for 3 h more. Postincubation, the media was collected, centrifuged at 1000 × g for 5 min to precipitate out any debris, and the resultant media was used for estimation of cytokine/chemokine release.