Culturing MDCK cells

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

1. Heat DMEM and trypsin in water bath
2. Discard old media with pasteur pipette and vacuum pump, while pointing flask towards flow and tilting it slightly to avoid touching cells.
3. Wash cells twice with ~2mL PBS per 25cm², discarding with vacuum pump.
4. Add 0.5-1mL trypsin per 25cm² and incubate for ~5min.
5. 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.
6. Add media (~3 times the amount of trypsin), and pipette up and down ~10 times to split cell clusters.
7. Move solution with cells to 15mL falcon tube and centrifuge. 500rpm for 3min.
8. Remove trypsin-media mixture with pipette (not vacuum pump!).
9. Add 6 mL of fresh media and vortex for ~30 sec.
10. Take 0.5 mL of cell mixture and add to new flask.
11. Write name, date, cell type and passage number on flask and incubate.
12. Clean up everything.