Section7:Typical Cell Culture Protocol

From Assay Guidance Wiki

1. Place media and trypsin in water bath (37°C).
2. Take flask of cells out of incubator.
3. Examine cells under microscope to determine health/condition.
4. In hood, remove medium from flask by aspiration.
5. Wash cells once with 5-10 ml of Phosphate Buffered Saline.
6. Aspirate off PBS.
7. Add 5-10ml trypsin or dissociation solution to cells.
8. Rock flask to allow above to cover cells.
9. Incubate 2-5 minutes or until cells begin sloughing off the flask.
10. Tap flask to see if cells have released from bottom of flask.
    • If not, incubate further 1-2 minutes or place flask in 37° incubator for few more minutes.
    • If so, go to 11.
11. Add equal volume of medium to cells.
12. Pipet up and down several times to break up cell clumps.
13. Pool all cell into one container.
14. Take out 1ml sample to count.
15. Count on hemacytometer, or automated cell counters like a Coulter Counter or Vi-CELL.
16. Do calculations to determine cell density.
17. Calculate amount of pool needed for desired cell number for project.
18. Take off that amount of cells and centrifuge at 1000rpm for 5+minutes. Determine amount media to resuspend cell pellet in.
19. Resuspend pellet in appropriate amount of medium.
20. Seed or plate as needed. Automated dispensers for cell plating will decrease variablility of cell seeding in 96, 384 or 1536 plates.