I've separated fibroblasts from mouse lung tissue, and put them in culture. After a month of being in culture, I noticed that they turned black, just like B16 melanoma cells. Do you have any idea if it is normal or not? Or they have been mixed with other cells and now they are not pure anymore?
I dont know what is the reason for fibroblast color...i wounder if you can send me the method for isolation of fibroblasts I'm eager to try performing it :)
all best
Nikola
It sounds very likely that they are contaminated. Black usually means melanin, and if you are using B16 in your lab then you might have cross-contaminated them. Try staining for melanoma antigens.
Yes! Finally I found out that they were contaminated. Thanks for your help.
And about the protocol, I cutting the lung, I put them in collgenase and DNase in 37°C for 45min. Then mesh through cell strainer and spin. If you put the cells in culture, after several days, the adherent cells will be lung macrophages and lung fibroblast. Using anti-CD11b antibody, you can easily sort fibroblast out of macrophages.
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