Remove all cell culture media
Wash the culture dish with 4ml sterile PBS (for a T-25)
Add 1ml Trypsin for T-25 and 2ml for T-75, incubate at 37°C for 5 min
Add 15ml of fresh cell culture media and wash the dish by pipetting
Collect the cells in 15ml labeled tubes
Centrifuge cells at 700 rpm for 10 min
Check for pellet
Resuspend the pellet in 0.5 ml freezing media for T-25 and 1.5 ml for T-75
Pipette into cryotubes, one for T-25 flask and 2 for T-75
Cell freezing:
Freezing media: 10% DMSO / FBS
Maximum 1,000,000 cells /ml freezing media / ampoule
Place the ampoules in the Styrofoam rack in –80°C over night,
Transfer to –140°C the next day
500 ml fibroblast Cell media contains
DMEM 425 ml
FBS 75 ml
L-glut 5 ml
Pen Strep 5ml