Splitting and Passaging Cells in Tissue Culture

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Splitting and Passaging Cells in Tissue Culture

1. warm media and the Trypsin-EDTA in the water bath (Trypsin-EDTA made by diluting the stock 1/10 by adding PBS only)
1. split cells when they’re about 80-90% confluent
2. wash cells with ~7mL PBS-EDTA (1mM EDTA)
3. aspirate off the PBS-EDTA
4. +1mL trypsin per 100mm dish
5. incubate in the 37-degree C incubator for 1-5 minutes (usually 1 min.); don’t do it much longer as it will damage the cells
6. take out dish from incubator and bang the sides of the dish all around
7. take a long pipet and pipet the solution up and down to break up any cell clumps (if it is absolutely crucial to get them broken up for cell counting or limiting dilution for cloning out into single clones, you may look under the microscope to see if all the cell clumps have been broken)
8. place solution into a sterile plastic tube with media containing fbs in it (fbs inactivates the trypsin, which was why it had to be rinsed off with PBS-EDTA initially)—a final concentration of ~1% fbs should be enough to inactivate the trypsin
9. using a balancing tube to balance the tube with cells, spin for a few minutes (~2 minutes should be enough)
10. aspirate off the media
11. resuspend in desired amount of media (if suspending in 10mL media, first resuspend cells only in 1mL, then resuspend in the rest, as it is easier to break up clumps using a small volume of media)
12. dilute to the appropriate amount (usually about 1/10 for a few days) in new dishes containing new media