Jacobs:Protocol Subculturing 3T3 Fibroblasts

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A general protocol for subculturing cells


  • 3T3 Fibroblasts (ATCC)
  • 0.25% Trypsin/0.03% EDTA (Gibco #25200-056)
  • Complete medium: 450 ml DMEM (Gibco #11995065) + 50 ml Calf Serum (Gibco #16010159)
  • PBS
  • Water bath
  • Tissue culture dish
  • Serological Pipets
  • Pipet aid
  • Pipette tips
  • 200 μL, 1000 μL pipetter
  • 15 ml, 50 ml Falcon tubes
  • Transfer pipet
  • Trypan blue
  • Hemocytometer, coverslip
  • Microcentrifuge tubes
  • Centrifuge
  • Cell counter
  • Timer
  • Waste beaker
  • 70% ethanol
  • Kimwipes
  • Markers
  • Gloves


  1. Heat complete medium, trypsin and PBS to 37ºC in water bath.
  2. Clean hood with 70% ethanol and bring materials inside hood.
  3. From incubator, remove culture dish and place into hood. Remove and discard culture media from dish using serological pipet.
  4. Wash cells by pipetting 8 ml of PBS in each culture dish. Remove PBS.
  5. Repeat PBS wash.
  6. Add 3 ml of trypsin/EDTA to each dish (for 100 mm dish) and return dish to incubator for approximately 5 min. Look under microscope to check detachment of cells. Tap side of dish if necessary.
  7. Once cells are detached, add 8-10 ml of complete medium to each dish to neutralize trypsin. Close lid and gently swirl medium.
  8. Pipet entire solution/cells into 50 ml Falcon tube.
  9. Centrifuge at 1000 rpm for 5 min. Remember to balance weight.
  10. Carefully remove supernatant without disturbing the cell pellet using transfer pipet and pipetter.
  11. Resuspend cells in 2 ml complete medium.
  12. Transfer 50 l medium/cell into microfuge tube.
  13. Add 50 l trypan blue to microfuge tube (dilution factor=2). Suspend well.
  14. Place coverslip over hemocytometer and using same pipetter, add enough solution to fill hemocytometer.
  15. Count cells under microscope. Total number of cells = count x 10,000 (hemocytometer factor) x 2 (dilution factor) x 2 ml (total volume)
  16. Add complete media to 50 ml Falcon tube such that each new plate can receive 1 ml of cells. For example, if the total cell count is 2,000,000 and you wish to seed 200,000 per dish (i.e., split ratio of 1:10), add 8 ml (8.05 ml to be exact since there should be 1.95 ml already in the tube)of complete media to the 50 ml tube to bring up to 10 ml of complete media. Mix well and seed 1 ml of cells per dish.
  17. Add additional complete media to each dish to make 10 ml.
  18. Look under microscope to monitor cells and return dish to incubator.
  19. Clean working space and dispose of biohazard waste.


  • Originally prepared by CRJ-CHK 1/16/04

or instead, discuss this protocol.