Passaging McCoy cells
We routinely use McCoy cells (supplied by the NCTC) for our cell culture work. However, this protocol is also used for HEp2 and BGM cell lines.
You will need:
- TE buffer (Trypsin/EDTA) ~3ml frozen aliquot (store at -20oC)
- DMEM (we buy ours from Invitrogen) + Glucose (store at 4oC) with 10% FCS added
- Tissue culture flasks
Protocol
- Take TE buffer out of freezer and DMEM out of fridge and allow to reach room temperature
- Take flask of sub-confluent cells (80-90%) from CO2 incubator
- Wipe surfaces, gloves and necks of all bottles with 70% IMS
- Pour medium off of cells
- Wash cells twice with cell culture PBS
- Add TE buffer to the flask and make sure it covers the whole monolayer of cells
- Pour off excess TE
- Lay flask flat and incubate for 5 minutes at room temperature for McCoy cells (or at 37oC for HEp2 cells).
- Tap side of flask to detach cells (should be easily detached after incubation)
- Add appropriate amount of DMEM, depending on requirements. For example, if you want cells the next day, add 3ml of DMEM and pipette up and down 3 times to break up aggregates.
- Add 20ml of DMEM to a new tissue culture flask
- Take 1ml of cells and add to the new flask
- Mix gently, loosen lid and place in 37oC / 5%CO2 incubator over night.
Sub-confluent McCoy cells, ready for passaging:
