Papain:

for 20mL in Hepes buffered Hanks salt solution

400uL papain

0.4g cysteine

pH to 7.4 with 1M NaOH

sterile filter

trypsin inhibitor:

for 12mL in Hepes buffered Hanks

0.12g Trypsin inhibitor

0.12g BSA

culture medium:

Either –

BME

5% calf serum

P/S, glutamine (1:100 of stock solutions)

3g/500mL of glucose

or-

neurobasal medium

B27 (50X) – 10mL in 500

glutamine, P/S

PLL or PDL coated coverslips (Sigma cell culture catalog)

Take isolated cotices in Hanks

Place in 10mL papain solution, 5’ at 37 degrees

Pipette off, and repeat

Pipette off and replace with 4mL trypsin inhibitor solution

2’ at RT

repeat 3X

resuspend cells in serum-containing medium and mechanically dissociate

for cell staining plate about 50,000/well of a 24 well plate