Primary cortical neuronal culture

For Primary cortical neuronal 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 (1500rpm,0h 0m 0s, 0h 5m 0s) and resuspend the pellet in 10ml medium supplemented with 10%

Triturate the cell suspension 10 times with a 1ml pipette

Coat 24 well plate with (PLL)

Place the cover glass at the bottom of the 24-well plate and add 400ul/well of PLL coating ( 0.01%) for 24h and incubate at 37°C

Wash 3 times with PBS after 24h 0m 0s

Seed the cells onto PLL coated 24-well plates at a cell density of 10⁶ cells/well containing specialized supplemented with 2%

Incubate for 24h 0m 0s at 37°C

Post 24 h, add 2µg/well of cytarabine (Stock - 5µg/ml) to the culture to inhibit the glial cell growth.

48h 0m 0s later remove the medium completely

Add 400µl/well of NB-A supplemented with 2% B27

Transduction with BRAF (Optional)

After 5 days of culture, these cells are ready for further experiment

After 5 days, the neurons were transduced with BRAFV^600E, or BRAF^WT, or vector lentivirus with 8 μg/ml polybrene (Sigma–Aldrich, USA) for 24 h.

The cells were cultured in NB-A for 120 h and used for subsequent experiments.