10/4/00
1. Cells need to be kept from becoming confluent. Check each day to make sure the cells are well spread out, flat, and not becoming crowded and cube-shaped. In general, a 1:20 split should last about 5-7 days.
2. When the cells are ready to be split, begin by warming up the medium, a bottle of calcium-magnesium free PBS, and a tube of trypsin. All medium should warm for about 20 minutes before using. Spray the bottles with ethanol before placing in the hood, and wear gloves.
3. In the tissue culture hood, aspirate off the medium from the plate to be split.
4. Wash the cells once with 5-10mL calcium-magnesium free PBS.
5. Add 1-3mL trypsin (just enough to cover the plate). Put the cells back in the incubator for 2-3 minutes. Check in the scope that the cells are coming up off the bottom of the dish before proceeding.
6. Once the cells have come up, add 10mL medium to the plate, and pipette into a tube.
7. Spin down the cells in the clinical centrifuge at 2000RPM for 5 minutes.
8. Aspirate off the medium, making sure not to suck up the cells.
9. Resuspend the cell pellet in 10mL medium.
10. Place a drop of the resuspended cells on the hemacytometer to count. You count the cells over the 5x5 square then multiply by 10 to the 4th to get the number of cells per mL.
11. Plate as needed for upcoming procedures or dilute 1:5, 1:10, and 1:20 for carrying.
12. Return cells to 10% CO2 incubator.
Medium:
DMEM (500mL)
10% Hyclone FBS (50mL)
Pen/strep, P/S (5mL)
glutamine, Q (5mL)