Splitting Cells

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

1. Warm PBS and Media in water bath
2. Wash cells twice with 10 mL (per 10 cm dish) PBS -/-
3. Add 1 mL trypsin and sit in the hood
4. Add 10 mL media to each new dish
5. Check cells for trypsinization, and if necessary tap the cells
6. Add 9 mL media to trypsinized cells
7. Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X)
8. 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