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.