Splitting Cells & Seeding Plates

STEPS: (WELL PLATES SHOULD BE SEEDED ONE DAY PRIOR TO TRANSFECTING)

1. Prior to transfection, the cells should be 70-90% confluent
2. Aspirate off the media from the dish the cells are growing in
3. Add 3mL of PBS/versene, aspirate
4. Add 2.5 mL of trypsin
5. Incubate for 2 minutes maximum (do not over trypsinize)
6. Add 9.5mL of complete media to inhibit the trypsin
7. Resuspend in a conical tube
8. Spin down in the centrifuge at 2500 RPM for 5 minutes at room temperature (20-25 C)
9. Aspirate off the media (be sure not to aspirate the pellet at the bottom!) and resuspend in 10mL of complete media.
10. Pipette 10-15 uL of culture from the conical tube onto a hemocytometer
11. Take the hemocytometer to the microscope and count the number of cells in a 4X4 grid (or take an average from multiple 4X4 grids)
12. The number of cells you count (if you are looking at a large 4X4 grid) is the amount per 0.0001 mL (every 100 cells you count = 1 million cells/ MILLI LITER)
13. Dilute a sample of the suspension to the correct cell concentration
    1. A new culture plate needs 1 million cells in 10-12 mL media
    2. A 24 well plate needs ~50,000 (attractene) or ~100,000 (Lipofectamine) cells in ~0.5mL media per well.
14. Pipette the necessary volume into each well of your plate for however many wells you need
15. Tilt/swirl to evenly distribute cells within the wells.

For Ramos cells:

Use modified RPMI-1640 media (it has some added components - the bottle has Brian's initials on it and it's on a shelf in the fridge).

1. Pipette cells from plate into 50mL conical tube.
2. Centrifuge at 2500 RPM for 5 minutes at room temperature.
3. Aspirate off media and resuspend in 10mL Ramos media.
4. Add 200,000 cells to about 10mL media in a plate (roughly 1:10 split every 3 days).