Splitting

  • Put 5 mL medium in each flask. Label with cell type and passage number
  • Warm flasks in incubator
  • Remove medium from cells.
  • Rinse once with ~3 mL sterile PBS. (Squirt in, then suck out.)
  • Add 15 drops sterile trypsin (=~0.75 mL)
  • Incubate (37C) 5 min
  • Check to make sure you can see them floating in the liquid
  • Add ~1mL medium to flask, draw up and down to mix.
  • Put
    • ~4-8 drops (= 0.2 – 0.4 mL) into each new flask containing medium
    • ~4-5 drops ( = 0.2 – 0.25 mL) if cells are 70 – 80% confluent
  • Put back in incubator.