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. 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 normal and high confluency
  2. Wash with 10mL PBS, swish around, aspirate and 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 prefer being stored in vapor as opposed to submerged in LN2.