Altman:Protocols/TC Protocols/Splitting ARPE-19: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
David Altman (talk | contribs) (New page: {{Template:Altman}} <div style="padding: 10px; width: 700px; border: 5px solid #B22222;"> ==Freezing down SF9 cells for long-term storage== 1. Remove the cell media from the cells 2. W...) |
David Altman (talk | contribs) No edit summary |
||
| (2 intermediate revisions by the same user not shown) | |||
| Line 3: | Line 3: | ||
<div style="padding: 10px; width: 700px; border: 5px solid #B22222;"> | <div style="padding: 10px; width: 700px; border: 5px solid #B22222;"> | ||
== | ==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 | 1. Remove the cell media from the cells | ||
| Line 9: | Line 15: | ||
2. Wash the cells with 5 mL of PBS | 2. Wash the cells with 5 mL of PBS | ||
3. Remove PBS, and put in 5 mL of Trypsin | 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 | ||
| Line 26: | Line 32: | ||
11. Re-suspend in 10 mL of media | 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 | |||
Latest revision as of 18:56, 4 September 2013
Department of Physics, Willamette University
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
