Altman:Protocols/TC Protocols/Splitting ARPE-19

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==Freezing down SF9 cells for long-term storage==
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==Splitting ARPE-19 cells==
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Before starting:
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* Warm up Media+Serum, PBS, Trypsin-EDTA, and FBS in 37°C water-bath
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* Place a beaker for disposing of waste in the hood
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* Place appropriate T-25 Flasks and 6 well plates in the hood
1. Remove the cell media from the cells
1. Remove the cell media from the cells
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11. Re-suspend in 10 mL of media
11. Re-suspend in 10 mL of media
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Adding cells to a T-25 flask:
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* 2 mL cells + 3 mL media (add media to the flask first)
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Adding cells to a 6-well plate:
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* Combine 4.5 mL cells + 4.5 mL media in a 15 mL conical
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* Put 1.5 mL into each well

Revision as of 21:53, 4 September 2013


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

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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