I work with B16-F10 and SK-MEL28 melanoma cells and I want to measure extracellular and intracellular melanin but I found different protocols and I have some questions. To measure extracellular melanin, I take some microlitres of culture medium (DMEM with phenol red + 10% SFB, glutamine and antibiotics) and I measure absorbance directly (I don´t do any procedure to the media); but I don´t know if the phenol red that it contains could interfere in the measurement. And to measure intracellular melanin, I lyse the cells in NaOH 1N during 45 minutes at 80°C (the volume I use of NaOH depends on the number of cells?). But I don´t have a positive control (like pure melanin or something like that) and I found in papers different wavelength to measure absorbance (415nm, 475, 472, etc) so I don´t know if I measure real melanin (I am using as positive control B16-F10 cells in confluency, and as negative control non-pigmented cells like colon cancer cells). Has anyone a protocol that could be useful? Thanks!!
Measuring OD is uselss unless you have a standard curve for comparison. You could use commercial synthetic melanin (https://www.sigmaaldrich.com/catalog/product/sigma/m8631?lang=en®ion=US) to generate a standard curve. It may not/prob does not - offer a precise weight but it would allow you to compare treatment and controls.
Hello Florencia
First and foremost phenol red in the culture medium will not interfere in the measurement as it shows strong absorption at wavelengths of 450nm and 560nm. Melanin absorbs visible radiation through two mechanism 1) entire visible range which is linear in wavelength and 2) wavelengths in the range of 400-500nm which is exponential in frequency. Melanin absorption maximum is 335nm. You can use 400nm as the wavelength to measure absorbance for melanin as other substances will not interfere in this range.
Secondly, prepare a standard curve for melanin using commercially available melanin in order to measure the extracellular and intracellular melanin content.
Thirdly, melanin content can be measured in two ways:
a) Melanin content per cell which is the quantity of melanin in each pigment cell
OR
b)Melanin content per area which is melanin per pigment cell and the cell density in a given area.
Hope this is helpful.
Thank you.
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