Altman:Protocols/TC Protocols/Splitting ARPE-19

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Current revision (21:56, 4 September 2013) (view source)
 
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2. Wash the cells with 5 mL of PBS
2. Wash the cells with 5 mL of PBS
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3. Remove PBS, and put in 5 mL of Trypsin
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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
4. Incubate for 10 minutes at 37° and 5% CO2

Current revision


Department of Physics, Willamette University

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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
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