Cell Passaging Protocol
Materials

Ethanol

Pasteur Pipettes

5 ml, 10 ml sterile pipets

Confluent cells in flask

Media with 10% serum and penn-strep

Trypsin

PBS

15 ml centrifuge tubes

Pipet Aid
Safety

Nitrile gloves

Safety glasses

Lab coat

Keep ethanol away from open flame
Procedures
1. Look at flask under light microscope to make sure around 90-100% confluency
2. Warm up all media, trypsin, PBS, etc in water bath for ~ 15 mins
3. Follow sterile technique
4. Aspirate media from flask
5. Add PBS to flask a. About 0.2 ml per cm 2 area
6. Aspirate PBS
7. Add trypsin to flask a. About 0.1 ml per cm 2 area b. Put cap back on c. Put flask in incubator for ~5 mins d.
Be careful, you don’t want cells in trypsin for too long
8. Remove from incubator
9. Tap flask on hard surface to detach cells
10. Look at cells under light microscope to ensure detachment of all cells
11. Add media to flask to neutralize trypsin a. About 0.1 ml per cm 2 area b. Or same volume as trypsin added
12. Pipet solution to a 15 ml centrifuge tube
13. Centrifuge tube at 1500 RPM for 5 mins a. REMEMBER TO BALANCE CENTRIFUGE b. Have a centrifuge with the same volume of water c. Place balance on the opposite location of cell solution tube
14. Aspirate supernatant from tube a. Make sure not to disturb cell pellet

b.
DON’T ASPIRATE CELLS
15. Resuspend cell pellet in media a. Use a p1000 pipet to ensure cells are not clumped b. Try to avoid bubbles
16. Take 10
 l of cell solution in p20 pipet
17. Add to hemacytometer
18. Follow hemacytometer protocol
19. Determine amount of cells you need to add for experiment
20. Replate cells for future experiments
21. Take a new flask
22. Label a. For example, John Doe or Jane Doe will label his/her flask with b. Initials, date cells added, cell type, passage number c. Initials = JD; date cells added = 10/31/14, cell type = ADSCs, passage number = P4
23. Add fresh media to flask a. About 0.2 ml per cm 2 area
24. Add appropriate dilution of cell solution a. For example, 1:2 dilution is half of your cell solution b. Depending on dilution, you have to check to make sure cells are not overconfluent c. Here are approximations of when you need to passage again depending on dilution
Dilution
1:2
1:4
1:8
1:10
1:12
1:16
Approximate time until next passage
1-2 days
3-5 days
5-7 days
6-8 days
7-9 days
8-10 days

25. Place flask in incubator
26. Exchange media every 2-3 days