Splitting cells

We normally split one confluent T-75 flask into a fresh T-75 flask.


  • Warm up media, trypsin-EDTA (optional PBS without Ca++ and mg++) and in 37o C bead bath.
  • Inside the hood, add 15 ml of media to NEW T-75 flask. (For T175 or T150 use ~ 40ml)
  • Aspirate media from cell culture flask using an aspirating pipette (No Cotton), wash once with 3 ml trypsin-EDTA solution. Rock flask to rinse monolayer.
  • Aspirate and add 2 ml Trypsin-EDTA solution. Rock flask.
  • Incubate at 37oC (or room temp) for 1-2 minutes or until cells begin to come off from the plate (look under microscope to see cells detach from the plate)
  • Stop trypsinization reaction by adding 8 mL of media to flask.
  • Pipet up and down gently several times to mix and break up clumps (otherwise cells will cluster together when plated).
  • Split flask 1/3 – 1/5 [example 10ml (1/5), remove 2ml and add to new flask].
  • Mix and look at flask (observe cells) under microscope
  • Close media bottle caps tightly and return to 4o C
  • Rinse hood surface. 70% ethanol is good except when using human, monkey and primate cell lines these require with 10% bleach (Must be made fresh). Dry surface.
  • Rinse hood surface with water. Dry surface.
    • Note This step is very important to remove bleach, it will corrode the surface.
  • Rinse hood surface with 70% reagent alcohol. Dry surface.

Split flasks ~2X per week. Adjust split dilution to accommodate.

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