Begin by dissecting the hippocampus from an embryonic mouse brain and place the dissected tissue in ice cold calcium and magnesium-free buffer or CMF. Next, remove the CMF and rinse the tissue four times with fresh cold CMF, swirling the tube gently in between rinses. Then add filtered 0.125%trypsin solution and incubate for 15 minutes at 37 degrees Celsius, swirling every five minutes.
After incubation, remove the trypsin solution and wash two times with ice cold CMF. Then add three milliliters of trypsin inactivation solution. Next, to dissociate the cells, titrate 30 times using two second draws with a 14-gauge needle attached to a three milliliter syringe.
Continue titration with two second draws 30 times using a 15-gauge needle, then 20 times using a 16-gauge needle, followed by 20 times with an 18-gauge needle. Then perform three second draws using a 21-gauge needle 15 times to complete cell dissociation. Check for cell clumps by placing a droplet of the cell suspension on a microscope slide.
Next, filter five milliliters of FBS using a 0.22 micron syringe filter. Gently layer the cell suspension onto the FBS in a 15 milliliter conical tube. Centrifuge the cells at 200 x g at four degrees Celsius for five minutes.
Remove FBS and resuspend the pellet in one milliliter of warm antibiotic antibiotic-free neurobasal media. Dilute the cell suspension in 0.4%trypan blue and obtain a cell count. Plate the cells in 750 microliters of antibiotic-free neurobasal media in a prewarmed poly-D-lysine coated 4-well chamber slide.
Incubate the cells in a humidity controlled chamber at 37 degrees Celsius and 5%CO2.
