Protocol for Subculturing NIH3T3-47 Cell Line

Protocol

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.