I have been culturing these cells for around 2 weeks and have passed them twice. They never reached confluency. I am culturing them in DMEM/F12 media + 10% NCS (Sigma) + 2 mM L-Glutamine (already in DMEM media). I have heard that these cells should grow like weeds (?), however I am not observing this at all! Any advice would be greatly appreciated.
Hi .
NIH3T3 usually growing fast, cells will be double every 18 hours, Use DMEM formulated with 10% FBS +antibiotic. However, in culture, cells should not be allowed to become completely confluent, If cells 80% confluent - split the cells:
Remove medium and rinse with Trypsin-EDTA -Add an additional 1-2 mL of Trypsin-EDTA and allow flask to sit at room temperature until cells begin to disperse (round up). Dispersal can be encouraged by incubating and discard the Trypsin-EDTA.
Add fresh culture medium to cells and aspirate and dispense suspended cells into new culture flasks or plates
Subculture ever 3 days. Renew medium 2 times a week.
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