It is important to bank and cryopreserve sf9 and HEK cell stocks. This is a relatively simple process, but materials should be prepared in advance, and stock counts should be as low as possible.

Cryopreservation of HEK293GnTI-

Materials Required

  1. DMEM/F-12 supplemented with 10% (v/v) FBS
  2. Freezing Container
  3. Tissue Culture (or other) high grade DMSO
  4. Cryogenic storage tubes
  5. Freezing Medium
    1. DMEM/F12/10% (v/v) FBS supplemented with 10% (v/v) DMSO: Prepare immediately prior to use. Will need 10mL. To prepare remove 9mL of DMEM/F12/FBS. Add 1mL of DMSO. Mix thoroughly.

Protocol

Cells should be fully confluent and healthy, and as early a passage as possible. Prepare freezing medium immediately before use. Label 8-10 cryovials before use.

  1. Culture cells to high confluency (85-90% is ideal)
  2. Wash with 10mL PBS, swish around, aspirate, then discard
  3. Add 2mL Trypsin-EDTA. Let sit on plate for 1 minute.
  4. Add 10mL of DMEM/F12. Tap aggressively to dislodge cells into fresh DMEM.
  5. Spin cells down for 3’/25C/1K rpm
  6. Decant and remove DMEM+Trypsin. Add 10mL of freezing medium and resuspend thoroughly.
  7. Dispense aliquots into cryotubes.
  8. Incubate in freezing apparatus in -80C. Let set overnight.
  9. Transfer tubes to liquid nitrogen storage dewar. Stocks should be preferentially stored in vapor. Liquid submersion is acceptable, however it should be acknowledged that this can weaken stock viability and has the potential to introduce contaminants to the glycerol stock.