Dear Mouchandra Paul

After several passages in culture, B16-F10 shows attenuated pigment production. When B16-F10 is grown in high-tyrosine containing media like DMEM, the high tyrosine content in DMEM promotes an early phenotypic drift towards either a mesenchymal-like or senescence-like phenotype and prevents the establishment of cultures of melanoma cells harboring differentiated features. On the other hand, when B16-F10 is grown in low tyrosine containing media, the differentiated melanoma cell culture retains its phenotype.

So, if B16-F10 is allowed to grow for several passages in DMEM (high tyrosine containing media), initially you will observe pigmentation, but later the pigment formation stops as in your case. Such cells grow in culture with subsequent passages, and there will come a stage when you will have a culture of B16-F10 which is not able to make any pigment (amelanotic).

So, to solve this problem, I would suggest you order another vial of B16-F10 from NCCS, Pune of an early passage (below 10). Culture the cells in F10 HAM (low tyrosine containing media) +10% FBS. This is a non-melanizing media. Whenever you need these cells to secrete the pigment, switch over to DMEM media. It works well. In this way, you may induce pigmentation in B16-F10 whenever you wish to.

Please note that pigment synthesis by B16-F10 melanoma cells is very sensitive to culture conditions. Growing these cells up to passage 28 in DMEM (high tyrosine containing media) will affect pigmentation. The detailed mechanism of how melanin content of B16-F10 cells decreases with passage number is provided in the articles attached below.

Article Downregulation of NFAT2 promotes melanogenesis in B16 melanoma cells

Article Tyrosine-Dependent Phenotype Switching Occurs Early in Many ...

Best.

Thank you Malcolm Nobre for such a detailed explanation. I will contact NCCS Pune for another flask of lower passage no.