Resuscitating McCoy cells
You will need:
- 500ml DMEM medium + glucose
- 50ml Foetal Calf Serum (FCS)
- Frozen aliquot of McCoy cells
- 75cm2 Tissue culture flask (T75)
- Pasteur pipette
- Universal containers
Protocol:
- Take DMEM from fridge and warm to room temperature
- Take FCS from freezer and thaw to room temperature
- Take an ampoule of McCoy cells from freezer
- Clean surface of class II hood, gloves and necks of bottles with 70% IMS
- Add the FCS to the DMEM and mix gently
- Pipette 20ml of DMEM/FCS into a T75
- Thaw ampoule of McCoy cells quickly in hands with lid loosened slightly
- Add 10ml of DMEM/FCS to a universal tube
- Add cells slowly with a Pasteur pipette to the universal tube
- Centrifuge at 596 x g for 10 minutes
- Decant the medium
- Re-suspend pellet in 4ml of fresh medium and mix gently
- Add slowly to the T75 flask and mix gently
- Loosen lid and lay down flat in 37oC / 5% CO2 incubator; incubate for 48 hours.
- Once monolayer is just sub-confluent, cells are ready to passage.