Protocol for Subculturing NIH3T3-47 Cell Line


  1. Remove and discard the culture medium.
  2. Briefly rinse cells with 2 ml of 0.25% (w/v) Trypsin solution to remove all traces of serum that contains Trypsin inhibitor.
  3. Add 3 ml of 0.25% (w/v) Trypsin solution to flask and gently rock the flask so that the solution covers the surface.
  4. Incubate the flask at 37°C, 5% CO2 for 2-5 minutes.
  5. Add 0.3-0.5 ml of the trypsinized cell suspension to the new sterile flask containing 20 ml of complete growth medium and 800 µg/ml neomycin. For 75 cm2 flasks, use approximately 105 cells/flask and subculture every 3-4 days.
  6. Place the culture vessel in the 5% CO2, 37°C incubator.

    Note: To disperse "clumpy" cells, gently pipet the trypsinized cells 2-3 times before adding it to the new flask.