Hello,
we have recently purchased B16F1 (mouse melanoma) from ATCC. At first cells grew ok (used:Dmem from Sigma +10%FBS), but the medium was getting darker starting form next they after seeding. I read, that those cells excrete some melanin into the medium. So, is that normal? But after passage 3 (start adding antibiotic P/S 0.5x) cells look so sick, don't divide, just dying more (medium very dark). Please, share you experience on how to grow these cells. What medium (type,brand) do you use? What % FBS? Do you add antibiotic? Very appreciated!
Hello,
I used to DMEM medium (Sigma) complemented with 10% fetal bovine serum (FBS) (Sigma), 1% L-glutamine, and 1% penicillin-streptomycin (Sigma). It is normal to B16F1 cells excrete melanin.
How do you do the passages? Do you use tripsin or cell scraper? Some cells do not accept well the cell scraper and this lead to cell lysis and death.
The FBS is inativated? Because There are FBS that need to be inativated before use and if it is not this may lead to cell death.
Hello Emanoel, thank you for your reply! I don't add L-Glu, do you think it is the reason? I use commercial 0.25% Trypsin-EDTA for 3min at 37C. The FBS is heat-inactivated. When usually your medium is becoming dark(after seeding)?
Hi there, there are different formulations of DMEM from Sigma, which one are you using? Anyway, I strongly suggest you to check whether in yours L-Glu is already there, otherwise you should add it in. These cells are not able to produce this aa, as well as several other factors necessary for their proper growth, thus after being thawed, they start becoming sick. Melanin is normal in the medium, especially because the cells have been recently buyed from you. They will lose almost all the pigment after being splitted many times. However, never had issues by using RPMI 1640 GlutaMax with 10% FBS. Good luck, and keep me posted if you need further advices.
I usually culture B16F10 cells long-term in HAM's F10 medium (with 10% FBS ans strep./pen.). For standard culture Ham’s F10 medium is preferably used because of low concentration of tyrosine which decreases cell melanisation. In these non-melanizing conditions cells grow beautifully and then for the experiment I switch them to DMEM medium, so they will melanize - usually with 1 day preincubation in DMEM before experiment.
Thank you all for your advice and suggestions! I am really thankful! We were able to rescue our cells by switching to Gibco DMEM medium+1x L-Glu+ 1x Sodium Pyruvate. Now we even add 0.5 %P/S and they grow good with less cell death.
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