It's the first time I culture Caco-2 cells.
use 10%FBS DMEM medium，not add antibiotics. culture in 6cm dish.
I find cells grow slowly.After 96hours，the confluence is not reach 80% and a lot of cells float in the medium.I use microscope finding black dots around the cell,some of which seem to adhere to the cell .And cells can't confluence where the black dots exist.
The color of medium is red,but the medium is a little cloudy,I guess it's a contamination,but I don't know what it is.
Is it bacteria? someone tells me it's maybe secretion or debris of cell.
Thanks for help.
Hi Jiazheng,
If your medium is cloudy it is definitively a contamination. Start over with a fresh sample. if you absolutely need to rescue the contaminated culture because you do not a backup then you can try adding antibiotics (starts with pen/strep), at higher concentration usually used, to "clean-up" your cells - most of them will die but you may be able to isolate a clone and generate a new clean culture. this second option is far of being optimal for numerous reasons, and should only be used when there is absolutely no other options.
Hope this helps. Norbert
your description resembles a mycoplasma contamination. to confirm this contamiation, you must achieve a PCR using the supernatant of cell culture. you can find visual evidence by staining DNA with a fluorescent stain (see pic). No antibiotic can be used to clear this infection. If you finally find a mycoplasma, I advise to first clean thoroughly the laminar flow as safety weighing cabinet, incubators and lab, secondly start a new cell culture with a batch of cell bank of your lab and finally ensure that Good laboratory practices are followed.
Thank you very much.Norbert
Now I am sure it is a contamination.I wil thaw a new vital of Caco-2 cell.
I guess the black dot is the origin of contamination.I will add antibiotics to the medium to watch whether the black dots decrease.
Thank you Carolina.
You give me a new idea.we have DAPI in lab, can it be used to stain mycoplasma？
Could you give me some protocols？
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