Afra

• 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