Primary microglial culture

Primary microglial culture - Use C57BL/6J mice at embryonic day 17

Anesthetized pregnant mice (1% sodium pentobarbital, 80mg/kg), dissect their embryos and collect the cortex.

(Separate and remove the soft membrane and blood vessels, rinse the cerebral cortex in PBS, and use the ophthalmic scissor to cut pieces of the cortex)

Collect the cortices in PBS in a 50 ml tube on ice

(The 50 ml tube contains 30 ml of PBS) On ice

Transfer the cortices to 15 ml tubes containing 1.5 ml trypsin–EDTA (0.25%) and incubate it at 37°C for 0h 15m 0s Dissociate the cortices by triturating with a 10 mL serological pipette 10 – 15 times

Centrifuge the dissociated cortices (400x g,0h 0m 0s, 0h 5m 0s). Aspirate the media and resuspend the pellet in 5 ml

Count the cells and plate them in a density of 50,000 cells/cm²into PLL coated T-75 flask. Makeup the volume to 15 ml with the

Change the culture medium the next day followed by the addition of a fresh culture medium every 168h 0m 0s till 14 days of culture

Put the flask on a shaker for 6h 0m 0s at 37°C (The microglia grow as a monolayer on the top)

Collect the detached cells, centrifuge (400x g,0h 0m 0s, 0h 5m 0s) and resuspended in 4mL of

Count the cells and plate at a density of 50,000 cells/cm² in a 24 well plate

Incubate them at 37°C in 5% CO2. After 2h 0m 0s microglia usually attaches to the bottom of the plate.

The purity of the cells was monitored by staining with ionized calcium-binding adaptor molecule 1 (Iba1) antibody

(Microglia obtained by this method have a purity of 90%-95% and can be identified by immunofluorescence staining.

Transduction with BRAF (Optional)

The purified microglial cells were seeded at a density of 80% and transduced with BRAFV^600E, or BRAF^WT, or vector lentivirus with 8 μg/ml polybrene (Sigma–Aldrich, USA) for 24h 0m 0s.

After transduction, the cells were cultured in with or without for 120h 0m 0s , and the medium, as well as the cells, were collected for subsequent experiments.