prepare 6-well plate at least 1hr before use (2ml PBS/2%FBS +2µl Laminin per well)
Remove livers from mouse and wash liver tissue in 10ml PBS in 10cm plate.
transfer liver tissue to a new10cm plate, add 5ml PBS/2%FBS, and Chop these pieces with a 5 razor blade cutting object, and then add 5ml 2mg/ml collagenase/ dispase solution(UCSF CCF)(stock 2mg/ml ) , incubate for 30~40min at 37°C.
Sieve liquid through medical gauze into 50ml Falcon tube(usually use the 10ml from the plate plus 10ml PBS/2%FBS to wash the plate and tissue chunks with)
spin ( 4°C, 1000rpm, 5 min ) and resuspend in 10ml PBS/2%FBS and filter through 70µm filter followed by 45µm filter. Bring up to 5~10ml and spin again( RT, 1000rpm, 2 min ) .
resuspend in 5-10ml of 1×RBC Lysis butter, incubate on ice for 5-10min.
bring up to 5~10ml PBS/2%FBS and spin ( RT, 1000rpm, 2 min ) .
repeat step 7 for 2~3 times.
resuspend the cells with Tm1 medium, and aliquot into 6-well plate.
Reagents
Laminin: L2020-1MG, Store at -20°C, sigma
sterile cotton gauze pad : 12-Ply; 3 x 3 in, #22-415-468 , Fisherbrand