Hi i am using HepG2 cells recently i encountered a problem. HepG2 cells contains small dots which sometimes affect the growth of cells or sometimes don't affect as can seen in pictures. media become turbid. i digest 3 minutes with trypsin and keep 1:3 ratio for passaging. but after 48 hours of incubation cells become contaminated. if someone can help in this regard would be highly appreciated. thanks
I think it is mycoplasma contamination.
Do a mycoplasma test. 1%BSA for cell fixation, 0.1%triton for permeabilization. Then incubate cells with 0.4 microliter DAPI in 1XPBS. See the cells in a 100x fluorescent microscope. If mycoplasma contamination is there means dot blue colors will be there in between the cells.
I'm afraid that this might really be contamination. I observed similar spots on the HepG2 cells, when I had contamination. In addition, the cells' morphology looked really weird and also their nuclei in my case.
Do you observe other stuff, like does the incubator smell when you take out your cells? Does your media change color?
Do you use antibiotics in your media?
Also check for mycoplasma contamination as described above. I would also test all the components you use. In my case I had to change the media, as it was contaminated and also I had to thaw fresh cells.
I would also think its mycoplasma. You can also run a pcr, protocols are published. If positive, through everything, and clean the incubator thoroughly.
Doctor, your cells are probably unhappy! its seems like a fungal (yeast) contamination. But If you are sure it's not fungal/mycoplasmal contaminated, the 'dots' are most likely apoptotic blebs - a sure sign of high stress. Do a DAPI stain and see under a 66-100x lens to confirm. An additional suggestion is to spin down some of the spent media, and add back to the culture (usually split 1:3 or 1:4) at ~15%. I've used DMEM(high glucose)(Sigma)+10%FBS (Hyclone or Gibco)+5%Lifor serum replacement+pen-strep to grow these. They do look like c#@* initially out of LN, but recover in ~7-10 days
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