Stacey’s protocol for freezing down cultured mammalian cells (3/7/2014).
1. As when splitting cells: wash a confluent 10 cm plate with PBS, add 1 mL trypLE, place at 37 C for 5 min.
2. Gently suspend using 4 mL media (must contain FBS to quench trypsin) and transfer the 5 mL of cells to a 10 mL conical. Lightly pellet cells at 1000 RPM for 3 min.
3. Aspirate media off pelleted cells. Gently resuspend in 1 mL freezing media (10% DMSO (tissue culture grade) in FBS) that was been pre-warmed.
4. Transfer to cryo tubes that are well labeled with the location #, passage #, date, and a cell line descriptor. Place tubes in special freezing container (container must be at room temperature) that has isopropanol to the fill line. This is located in the tissue culture room and needs to be signed out. Place container at -80 C for at least overnight to allow the cells to freeze at a constant, slow rate to reach -80 C.
5. Transfer tubes to cryo-storage in the tissue culture room. Record all pertinent information (including location of tubes!) in the tissue culture record book located in the Berglund lab (above Leslie’s desk).