From OpenWetWare
Jump to navigationJump to search

<- Back to Protocols

Advancing Adherent Cells

Karmella Haynes & Ben Nyer, 2014

This protocol outlines the steps necessary for advancing a cell culture from a small flask (starter T-25 or medium T-75) to a larger flask (medium T-75 or large T-150). The steps are very similar to the Passaging Adherent Cells protocol, with the exception of volumes of reagents used.


  • 70% ethanol spray bottle
  • 1x Phosphate-buffered saline (PBS)
  • Trypsin-EDTA buffer/ media
  • Complete cell culture medium (e.g., 10% FBS, 1% pen-strep)
  • Sterile tissue culture-treated vent-capped T-75 flask or T-150 flask, one per new culture
  • 100% confluent (dense) cell culture (check confluency under the light microscope)
Three flask sizes: T-25 (small, for starting cultures from frozen stocks), T-75 (medium, for routine passaging and expanding cell cultures), and T-150 (large, for growing & harvesting large amounts of cells). A flask must be properly laid flat (where the opening tilts upward) so that the cells can adhere to & grow on the bottom inside surface.


NOTE: Volumes of reagents are in X//Y format. If you are transferring from a T-25 to T-75 flask, use the left values. If you are transferring from a T-75 to a T-150 flask, use the right values.

  1. Pre-warm all liquid reagents to 37°C in the bead bath.
  2. After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood.
  3. Retrieve the cell culture from the incubator. Stand the flask up (see image above). Open the cap and aspirate old culture media from the bottom of the flask.
  4. Add 2//5 mL 1x PBS to the flask. Lay the flask flat (see image above) and wash by gently tilting the flask back and forth.
  5. Stand the flask up. Open the cap and aspirate off the PBS.
  6. Add 1//2 mL trypsin-EDTA into the bottom of the culture flask. Lay the flask flat and coat the cells completely with the trypsin-EDTA. Let the flask sit in the hood at room temperature for 5 minutes.
  7. After incubation, examine the cells under a microscope. Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask.
  8. If the cells are not fully detached, place the flask back into the 37°C incubator. Some cells may require some mechanical agitation (including “tapping” the flask).
  9. Once the cells have detached, stand the flask up and add 4//8 mL serum-containing medium (e.g., 10% FBS) to the cells to deactivate the trypsin. Repeatedly suck up and dispense the medium from the pipette to "wash" cells off the growth surface and into the bottom of the flask. Leave the flask standing up.
  10. Get a new T-75 or T-150 flask, label it with the cell line name, your initials, and the date. Include the passage letter (e.g., PS-β if you are advancing from a T-25 to a T-75 flask, PS-γ if you are advancing from a T-75 to a T-150 flask).
  11. Stand the new flask up. Add 5//10 mL growth medium to the new flask. In the old flask, gently resuspend the cells by pipetting up and down about 5 times. Transfer the entire volume of the old flask into the medium in the new flask. Lay the flask flat and push it back-and-forth and side-to-side to spread the cells evenly. Do not swirl it in a circular motion.
  12. Place the new flask into the 37°C incubator. The cells should adhere to the growth surface in a couple of hours.
  13. Discard the left-over cells as liquid waste by aspirating them into the vacuum trap. Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste.