Splitting Cells

SOP

• SOP- Biosafety Cabinet
• SOP- Cryogenic Materials
• SOP- Vacuum Pumps

Materials

• Media (FBS or NCS as required): Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM

(To prepare DMEM: 1 bottle of DMEM found in 4c in cell culture room 1 tube FBS found in -80 in cell culture room 5 mLs PSG found in door of 4c in cell culture room 500 mL bottle taken from lab filter found on metal rack behind fume hood •pour all ingredients into DMEM bottle •screw filter onto 500 mL bottle •attach hose onto arm of filter •pour mixture into filter)

• PBS -/-
• 0.05% Trypsin

Protocol

1. Warm PBS and Media in water bath
2. Aspirate the plate media
3. Wash cells once with 10 mL (per 10 cm dish) PBS -/- then aspirate the PBS
4. Add 1 mL trypsin and allow to sit in the hood for 2-5 min
5. Add 10 mL media to each new dish
6. Check cells for trypsinization, and if necessary tap the cells
7. Add 9 mL media to trypsinized cells
8. Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X)
9. Replace plates in the 37C 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