Cell culturing protocol

Thawing Frozen Cells

1. Cells arrive deep-frozen.
2. Add cells to 10 mL of growth media with 20% FBS.
3. Incubate overnight or until confluent.
4. Passage to 2 dishes with 10 mL of growth media with 10% FBS
5. Continue to passage about twice a week.

Passaging (SCC7)

1. Aspirate (remove) media.
2. Add 1 mL of trypsin, coating the entire bottom of the dish.
3. Swirl for 20 seconds, then aspirate.
4. Add 1 mL trypsin.
5. Incubate for 4 minutes.
6. Use the pipette to “pressure wash” cells off of one dish with trypsin.
7. Add the trypsin with the lifted cells to the second dish.
8. Use pipette to wash cells off of the second dish with trypsin.
9. Add (x) μL of trypsin with lifted cells to new dishes with 10 mL of growth media.
   1. (x) should be somewhere between 75 and 150, depending on how quickly you want the cells to reach confluence

Passaging (HeLa)

1. Aspirate (remove) media.
2. Add 10 mL of HBSS or PBS.
3. Aspirate.
4. Add 2-4 mL trypsin.
5. Incubate for 3 minutes.
6. Use the pipette to “pressure wash” cells off of one dish with trypsin.
7. Add the trypsin with the lifted cells to the second dish.
8. Use pipette to wash cells off of the second dish with trypsin.
9. Add (x) μL of trypsin with lifted cells to new dishes with 10 mL of growth media.
   1. (x) should be somewhere between 75 and 150, depending on how quickly you want the cells to reach confluence