Cryopreservation of Mouse ES Cells
- Freezing vials (1 or 2 ml size)
- Styrofoam or iso-Propanol container
- -80° freezer
- Liquid Nitrogen Storage Tank
M15 or ES Cell Culture Media:
- 82% high Glucose DMEM (Invitrogen, catalog number: 12100-061)
- 15% Fetal Calf Serum (ES Cell Tested)
- 1% 200mM L-Glutamine (Invitrogen, catalog number: 25030-081)
- 1% Penicillin Streptomycin (Invitrogen, catalog number: 15140-122)
- 1% 10mM ß-mercaptoethanol (Sigma, catalog number: M 7522)
(7.5ul ß-mercaptoethanol + 10ml PBS)
ES Cell Freezing Media:
- 60% DMEM high Glucose (Invitrogen, catalog number: 12100-061)
- 20% FCS (ES Cell tested)
- 20% DMSO (Sigma, catalog number: D-2650)
Working Solution (1X Freezing Media):
- 1 part 2X freezing media + 1 part M15
- Feed cells 2 hours prior trypsin treatment.
- Rinse plate twice with PBS.
- Trypsinize cells for 6-10 minutes at 37°C.
- Place an equal volume of ES Cell Media as Trypsin and pipette vigorously in order to disrupt any cell clumps.
- Using ES Cell Media, bring volume up to 10mls and place in a sterile 15ml conical tube.
- Using a hemocytometer count cells to determine total number of cells in tube.
- Spin at 600rpm for 5 minutes to pellet cells.
- Resuspend cell pellet in enough freezing media to make a 1 x 107 cells per ml suspension.
- Place one ml cell suspension per cryovial.
- Allow to freeze overnight in Styrofoam container at -80°C.
- Place in LN2 storage only after overnight storage at -80°C.