Submitted by kdorfman on Mon, 07/09/2012 - 19:44
  • Put 5 mL medium in each flask. 2.5 ml for mini flasks. Label with cell type and passage number
  • Warm flasks in incubator
  • Remove medium from cells.
  • Right away, Rinse once with ~3 mL sterile PBS. (Squirt gently in, Rock the flask, and then suck out. Do not hit the place where cells are. )
  • Add 15 drops sterile trypsin (=~0.75 mL) - enough to cover cells, if more than a thin layer, pull some out
  • Incubate (37C) ~5 min or until all floating, likely no more than 5m.
  • Check to make sure you can see them floating in the liquid
  • Add ~1mL medium to flask, draw up and down to mix.1
    • ~4-8 drops (= 0.2 – 0.4 mL) into each new flask containing medium 23
    • ~4-5 drops ( = 0.2 – 0.25 mL) if cells are 70 – 80% confluent
  • Put back in incubator.


  1. Use a ratio of medium: trypsin of 2:1 if you need the cells to hang out longer (say for counting) before going into the new flask. ↩︎

  2. if original flask was totally confluent, then use ~1/10th to ~1/5th of the volume; use more if you need cells in a day or two, use less if you don't need cells for a few days. ↩︎

  3. For dishes: if you want a nice monolayer of cells to form within one or two days, add a drop or two of cells AND examine in the microscope. You can quickly get a feel for the number of rounded cells and how heavy the plating will be. You can measure the exact volume and keep track if that helps. Remember to rock to distribute cells on the coverslip or Mattek surface (glass bottom dish). Do not swirl. ↩︎