I am currently using a B16F10 (murine melanoma) cell line for my anti-tyrosinase experiments. However, there are times where I noticed that the same treatments (e.g., my compounds at the concentration of 250ug/ml) would produce vastly different results in terms of cellular tyrosinase activities and melanin productions.
Besides, there are also times where the same treatments would reduce the tyrosinase activity with no effect on melanin production on cells (e.g. in p5), and reduce the melanin production with no effect on tyrosinase activity on cells (p.g. in p6).
I've done multiple "exclusion" works such as amending the FBS (brands, lots, heat-inactivation, concentrations etc.), with and without antibiotics (PenStrep, Gentamicin, etc.), additives on medium (HEPES, L-glutamine), yet the performances of the cells still fluctuate at times.
Is there any special attentions or conditions that I need to adhere to? I would appreciate if colleagues who are currently working or have worked with B16F10 cells (or B16, B16F1, etc.) would kindly provide their insights or opinions of their works with the cells.
Melanin synthesis by B16 cells is highly dependent on local cell density and on the pH of the medium. To get consistent results you need to control both of those carefully.
pH: We grow these cells in a low-bicarbonate, low-tyrosine medium like RPMI 1640 or MEM with nonessential amino acids, plus FCS, in a 10% CO2 incubator which gives a neutral pH of 6.9 to 7.0. That reduces pigmentation versus say DMEM and/or 5% CO2. Media with higher pHs give higher pigmentation and lower growth. Tyrosine level of course also affects pigmentation.
Density: We count the cells and for normal stocks we plate at 1 x 10*4 cells/ml. It is important to get a single-cell suspension and plate very evenly. They will grow around 20-30x in 3 days, or ~60x in 4 days. They are subcultured 2x per week.
This kind of protocol gives very low pigmentation and fast growth. To increase pigmentation, you can increase the pH (extra bicarbonate), and/or add an inducer such as a cAMP agonist or MSH.
Don't let the cells get confluent - unless you change the medium daily. B16 cells are highly transformed and will try to grow, exhaust the nutrients, acidify the medium and many cells die.
I undertand you. I have seen fluctuations like that. Sometimes, inhibition of tyrosinase activity enhances the expression of the tyrosinase gene, On the other hand, TRP1 and TRP2 can affect melanin production without altering tyrosinase activity. It is difficult to control those fluctuations.
You could try, serum-free hormonally- supplemented medium if there is one already for these type of cells. If there is not, if you are interested, it coudl be a good idea to formulate one for them so that you may know exactly what you are adding and that there are no inhibitors of activators of the enzymes you are studying .
Melanin synthesis by B16 cells is highly dependent on local cell density and on the pH of the medium. To get consistent results you need to control both of those carefully.
pH: We grow these cells in a low-bicarbonate, low-tyrosine medium like RPMI 1640 or MEM with nonessential amino acids, plus FCS, in a 10% CO2 incubator which gives a neutral pH of 6.9 to 7.0. That reduces pigmentation versus say DMEM and/or 5% CO2. Media with higher pHs give higher pigmentation and lower growth. Tyrosine level of course also affects pigmentation.
Density: We count the cells and for normal stocks we plate at 1 x 10*4 cells/ml. It is important to get a single-cell suspension and plate very evenly. They will grow around 20-30x in 3 days, or ~60x in 4 days. They are subcultured 2x per week.
This kind of protocol gives very low pigmentation and fast growth. To increase pigmentation, you can increase the pH (extra bicarbonate), and/or add an inducer such as a cAMP agonist or MSH.
Don't let the cells get confluent - unless you change the medium daily. B16 cells are highly transformed and will try to grow, exhaust the nutrients, acidify the medium and many cells die.
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