Notes: Steps should be done inside of a Biological Safety Cabinet in Sterile conditions. Make sure that the area has been cleaned with 70% ethanol, the UV light is off, and that the blower has been on for at least 10 min before starting. Once finished close clean the area off with 70% ethanol, close the cabinet and turn on the UV light.
Procedures (done in T25 flask):
- Check under microscope to make sure that cells are less than 80% confluent
- Remove the solution from the flask
- Rinse with DPBS (Divalent free Dulbecco’s Phosphate Buffered Saline) ~ 5 mL
- Remove the solution
- Add 1 mL 0.05% Trypsin EDTA
- Place in 37° C incubator until cells detach from the dish ~ 5 min
- Check under the microscope to make sure cells have detached
- Add 5 mL of Cell Media into a new dishes
- Check under the microscope to make sure the cells are dispersed into single cells.
- Add the appropriate amount of cells into the new dishes
- Swirl the dish or pipet up and down
- Label the Dish: Name, Date, Cell Line, Passage #, Ratio
- Cell Media: Ham’s – F12 from Gibco, 10% FBS, Penicillin/Streptomycin
To make add 500 mL Media + 50 mL FBS