Culturing MDCK cells
From mn.fysikk.laglivlab
MDCK is usually cultured in T25 flasks, i.e. the amounts given here are for T25 flasks (25 cm²).
Equipment
- PBS
- Trypsin
- DMEM + 10% FBS + 1% P/S
Protocol
- Heat DMEM and trypsin in water bath
- Discard old media with pasteur pipette and vacuum pump, while pointing flask towards flow and tilting it slightly to avoid touching cells.
- Wash cells twice with ~2mL PBS per 25cm², discarding with vacuum pump.
- Add 0.5-1mL trypsin per 25cm² and incubate for ~5min.
- Check detachment percentage. If low, tap flask carefully and incubate longer. Do not leave for longer than 15-20min as trypsin is harmful to cells.
- Add media (~3 times the amount of trypsin), and pipette up and down ~10 times to split cell clusters.
- Move solution with cells to 15mL falcon tube and centrifuge. 500rpm for 3min.
- Remove trypsin-media mixture with pipette (not vacuum pump!).
- Add 6 mL of fresh media and vortex for ~30 sec.
- Take 0.5 mL of cell mixture and add to new flask.
- Write name, date, cell type and passage number on flask and incubate.
- Clean up everything.