Altman:Protocols/TC Protocols/Splitting ARPE-19

Department of Physics, Willamette University

'

Home        Contact        Lab Members        Publications        Research        Protocols        Pictures       

Splitting ARPE-19 cells

Before starting:

• Warm up Media+Serum, PBS, Trypsin-EDTA, and FBS in 37°C water-bath
• Place a beaker for disposing of waste in the hood
• Place appropriate T-25 Flasks and 6 well plates in the hood

1. Remove the cell media from the cells

2. Wash the cells with 5 mL of PBS

3. Remove PBS, and put in 5 mL of Trypsin (Trypsin .05%, Life Technologies, Cat. 25300054)

4. Incubate for 10 minutes at 37° and 5% CO2

5. Look at the cells using the microscope; you should see the cells detaching from the surface

6. Knock the flask to dislodge the cells

7. Put in 1 mL of FBS

8. Put the 6 mL of cells into a conical

9. Spin at 500 rpm for 3 minutes

10. Pull off the supernatant (6 mL)

11. Re-suspend in 10 mL of media

Adding cells to a T-25 flask:

• 2 mL cells + 3 mL media (add media to the flask first)

Adding cells to a 6-well plate:

• Combine 4.5 mL cells + 4.5 mL media in a 15 mL conical
• Put 1.5 mL into each well