I have been trying to proliferate cells for almost 4 weeks culturing 4T1 cells in RPMI 1640 medium supplemented as recommended by the ATCC (10% FBS, 5% antibiotic).
Started with a frozen vial (passage # 4).
Cells are not growing at all. I even increased FBS to 20% and have been changing medium every 2-3 days, passing cells 1:2 and can't see any sign that cells are getting off stationary phase.
Any recommendations?
I will appreciate your input. Thank you
Giuliana D Noratto came across a question and answer typically not same but somehow linked to your question so felt like sharing the answer and you can see how Dr S J Malik https://www.researchgate.net/post/Problem_with_4T1_Tumor_Model_Development-Can_anyone_help
This is for your refernce
Hello Giuliana D Noratto
You need to recheck the concentration of antibiotics used in the growth media. Excess antibiotics used in culture can harm the cells. They deeply influence cell metabolism. There has been evidence of anti-proliferative effects of beta-lactam antibiotics like penicillin on cultured eukaryotic cells. The cells show poor growth rate and adherence to the culture dish in the presence of excess antibiotics. Antibiotics can significantly alter the gene expression and regulation.
Usually, there is no need to add antibiotics to the culture if you maintain sterility following standard protocols. Even if you need to add antibiotics, we usually add 1% penicillin-streptomycin solution.
So, 5% antibiotics may be too high a concentration. This could possibly be the reason. Please check this out.
Best.
Hi Giuliana,
As Malcolm mentioned, the antibiotic may be the problem, we use 1% penicillin/streptomycin and the rest ingredient does not differ much from yours, therefore I suggest you may decrease the antibiotic concentration, which may solve the problem.