Hello everyone. In my experiment in 24wp some of the wells look like more fibroblast than hela. I used RPMI with 10% FBS and no antibiotics. Do I have a contamination? or are my cells dead? The medium is clear and I do not see any bacteria. I checked the stock that I used and it was fine. And the other wells looks fine too.Do you have any idea why are they like that and what should I do to avoid this. Thank you in advance!
Hi,
I think you do have a contamination, but not with microbes, but with another cell line in your HeLa-Cells.
There are (obviously by size) at least two distinct populations present (upper-right quadrant, much bigger than the other cells) in your culture, did you grow them from extremely low cell densities? Because usually, to get as spatially clearly separated populations as in your picture, you would technically have to bring the cells down to cloning concentrations (at most a few hundred cells per vessel) to get such quasi-monoclonal colonies, what I think has happened here.
Do you handle several lines in parallel in your lab? If so, compare the morphologies and size of those lines with those observed in your picture, my first guess is that you have contaminated your HeLa-Cells with another line. Another explanation is that there has been a spontaneously developed sub-population risen from "original" HeLa (e.g. tetraploids, which can explain the bigger size), however I recommend try to get fresh stocks, ideally from a reputable source, e.g. ATCC.
Hela cells does not have shape of fibroblast. If your you are getting fibroblast shape of cells in the culture flasks, then your should first check contamination on the basis of streak separately on the Nutrient agar and Potato dextrose agar plates separately for bacterial and fungal contamination. you observe cells containing culture flask color whether it red or turned into the yellow. if color is yellow, then might be possibility of contamination. if your are handling other cells lines together, which may get contaminated like NIH/3T3 which is a fibroblast cell lines.
I will suggest you to passage the cells and may order for new stock of the cells.
Regards
Dr. Khem
Thank you all for your kind replies. This weird population appeared only some wells. And the other types of cell lines that we work have no morphology like this. I showed the cells to my other colleagues and we couldnt figure it out why. I will check the viability of them via flow cytometer if I have another contaminated cell line I must see another population as far as I know.
I seed 15000 cells per well because i need this confluency for the experiment but now I have this weird population in my control well. I am not sure whether I can trust or not other wells even if they dont have such population.
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