I want to perform a time point analysis of expression of cytokines and signaling proteins in Jurkat T cells treated with a compound X. The confusion is whether these cells, being lymphomas have the characteristic of constitutive expression of inflammatory factors and thus will impact the interpretations of my results. Please let me know fellow researchers.
Hi there Anusree
It's an important question. I suggest that you look at the multi-cytokine immunoblots sold by RayBiotech. These will let you assess up to >100 different soluble products that your cells make constitutively and (for example) after stimulation with PMA or some other mitogen. This will give you a baseline of how your cells behave, in your hands, in your lab. We have used these successfully more than once:
http://www.raybiotech.com/cytokine-array.html
You might also paus to ask whether it's worth the extra investment to get a fresh ampule of Jurkats from ATCC or EACAC, to avoid problems that may result from the cells being contaminated with mycoplasma, for example, or in extreme, not being Jurkats at all....
Good luck!
G
Hi Anusree
The few times I did cytokine measurements with stimulated Jurkat, my negative control (media alone) showed no relevant cytokine levels. But once I had a problem with a cell culture that I had been cultivating for a long time (over 15 passages). I assayed IFNg, IL2, IL4 and IL10.
Hi Grant and Andre
Thank you for your valuable inputs. I would also like to ask if Jurkats have a high chance of mycoplasma contamination. I had revived the cell line from cryopreservation almost two weeks ago but the cells have still not grown confluent. Do you think it could be mycoplasma contamination or its just that it's taking time to get revived from cryopreservation? Thank you again.
Mycoplasma is a possibility. You could run a PCR using Mycoplasma primers to confirm it. The problem will be if the contamination happened before cryopreservation, then all your stock will be contaminated. Try a new vial or ask another lab for a cell vial.
what Andre said makes sense - it shouldn't take such a long time for the cells to recover.
Are you sure that there are any live cells? If you think that there are a lot of dead cells, then you can get rid of them by running the cells over ficol, just as if they were a blood sample. Dead cells will take up the ficol and sediment, while cells with an intact membrane will not enter the ficol and can be recovered from the interface.
Good Luck!
G
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