When we tried to treat endothelial cells with Sodium polyoxotungstate (POM-1) which is a NTDPase inhibitor, to our surprise, all treated cells were dead after o/n treatment. In the end we were able to conclude that this was because of the antibiotics used in Lonza´s EBM-2 endothelial cell culture medium (with bulletkit). Most likely the adverse effects were caused by Amphotericin B together with POM-1, since with different or with no antibiotics this problem was resolved.
This is more just a comment to warn others of trying this, than a question but it would be nice to put together similar stories if you would have some similar unexpected difficulties in your experiments.
While I was at Harvard a batch of VWR media was toxic for some reason i.e. without addition of anything else. It took me a while to figure out as the entire batch was affected but in the end we grew 3 lots of cells in 3 media and only the one from the recently delievered batch died. We tend to blame ourselves rather than our reagents. The company kindly offered a refund but we did waste a few weeks.
Some cells are sensitive to antibiotics as well. When I started to work with endothelial cells, I was using pen/strep in lower concentration (1/2) than usual. But with time I switched back to normal conc and had no problems.
FBS is important factor since it can vary from batch to batch, but i don't think this was the case in your story.
Thank you for sharing your experience. Jonathan is right - we tend to blame ourselves rather the reagents.
Good luck!
Amphotericin B can have profound effects on cells in culture. A few examples from my own experience:
1) Enhances response of early erythroid progenitors to erythropoietin.
2) Suppresses growth of granulocyte/macrophage progenitors.
3) Dramatically (up to 1000-fold) enhances the killing of tumor cells by Merocyanine 540-mediated photodynamic therapy. (See Tsujino et al, Photochem Photobiol, 82: 458-465 (2006) for details)
4) Enhances the binding of Merocyanine 540 to target cells.
My advice: Keep Amphotericin B out of tissue culture medium unless the puprose of your experiment is to study the effect of Amphotericin B.
I don't use any antibiotics and my cells (Hacat , JURKAT) are growing happily with no infection. In my opinion If you follow good practice, u don't need to add antibiotics. furthermore, Antibiotics might affect my genes of interest as I am doing qPCR analysis after treating them.
Thank you for your interesting stories and comments! I have also heard that culturing cells with antibiotics is unnatural but it is old habit to use some. And in vitro studies are in all case a bit unnatural. Since we are using expensive commercial primary cell lines and our laminar flow-hood is not working so great I guess I will move on to more traditional P/S antibiotics. Also, we use media with antibiotic for mostly cell stock cultures and usually not when we are doing an experiment, except now with POM-1, which did not work out so well.
I would avoid the routine use of antibiotics and this includes penicillin and streptomycin and all antifungals like amphotericin B. The only exceptions would be circumstances under which it is impossible to eliminate potential routes of contamination e.g. extraction of primary cells from animal or medical tissues. Once such procedures are complete it's best to work antibiotic free. Amphotericin B has some detergent like non-specific properties which can increase the toxicity of some compounds and alter the sensitivity of receptor mediated events.
I second Fatma and Jim about not using antibiotics. For Primary culture we never use antibiotics/antifungals. We prefer a clear contamination and discarding a few plates than a lingering low-intensity contamination that would go unnoticed but mess all the downstream experiments. I hope this helps
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