Difference between revisions of "Splitting Cells"
From Bridges Lab Protocols
Davebridges (Talk | contribs) m (→Cell Specific Notes) |
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#Warm PBS and Media in water bath | #Warm PBS and Media in water bath | ||
#Wash cells twice with 10 mL (per 10 cm dish) PBS -/- | #Wash cells twice with 10 mL (per 10 cm dish) PBS -/- | ||
| − | #Add 1 mL trypsin and sit in the hood | + | #Add 1 mL trypsin and sit in the hood for 2-5 min |
#Add 10 mL media to each new dish | #Add 10 mL media to each new dish | ||
#Check cells for trypsinization, and if necessary tap the cells | #Check cells for trypsinization, and if necessary tap the cells | ||
| Line 13: | Line 13: | ||
#Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X) | #Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X) | ||
#Replace plates in the incubator | #Replace plates in the incubator | ||
| + | |||
==Cell Specific Notes== | ==Cell Specific Notes== | ||
*3T3-L1 fibroblasts have special considerations regarding confluence. See [[Differentiation of 3T3-L1 Cells]] | *3T3-L1 fibroblasts have special considerations regarding confluence. See [[Differentiation of 3T3-L1 Cells]] | ||
Revision as of 17:29, 26 October 2009
Materials
- Media (L1-FBS for 3T3-L1, COS-FBS for others): Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM
- PBS -/-
- 0.05% Trypsin
Protocol
- Warm PBS and Media in water bath
- Wash cells twice with 10 mL (per 10 cm dish) PBS -/-
- Add 1 mL trypsin and sit in the hood for 2-5 min
- Add 10 mL media to each new dish
- Check cells for trypsinization, and if necessary tap the cells
- Add 9 mL media to trypsinized cells
- Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X)
- Replace plates in the incubator
Cell Specific Notes
- 3T3-L1 fibroblasts have special considerations regarding confluence. See Differentiation of 3T3-L1 Cells
- RAW 264.7 cells are scraped, not trypsinized. See Culturing RAW 264.7 Cells
- S2 cells are grown at 28C without extra CO2. See Culturing S2 Cells
