There is no color change in media. Black spots can't be removed by PBS washing.
Lakshmi,
From the images you posted, dots are not like contamination. Cells are in stress. could you please try to give some information,
1) the exact composition of medium you are using? sodium bicarbonate amount? pH? Non essential aminoacids? Antibiotics concentration? any additives(insulin/transferrin/glutamate)?
2)at what confluency you are spliting cells? High confluency >90% may give debris.
3) how much time you are incubating your cells with trypsin? For HepG2, 60mm dish, 80% confluency, 1-2min is enough. We have to use optimum trypsin, for optimum time for each cell line. Unless that gives clumps or debris.
4) What is the passage number of the cells(~)? The cells in image are not in same morphology, mixed population, may be higher passage or just revived.
In my experience the black dots are debris. If you place another image with higher resolution you will receive more help. Best regards
Thanks for your informations Mr. Chandra Shekar Dasari.
Yes, your cells are stressed and dying. Those dots are debris from dead cells, and I think just by the looks of those images its because of your subculturing. I think your seeding your flasks with not enough cells and they're dying. I agree with Chandra, we need to know more about the cells your culturing and how exactly your culturing them before we can give you a good idea on how to proceed
Even I think these are not contamination. The seeding density seems low. Most of the cells do not have proper morphology and lacking intracellular connections. Therefore most of the cells are spindle like; rest are glowing in clumps and are stressed.
Check seeding, splitting ratio optimum for this cell, media, serum, cell revival.
Good luck.
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