Hi,
A year ago, I did some ELISA experiments in the medium of cell culture of NK92 and got low IFN-gamma release (around 50pg/mL). It was nothing compared to my stimulated conditions where I got around 4000pg/mL.
Recently, I did again some ELISA and obtained a very high IFN-gamma background in my unstimulated conditions and sometimes this background is as high as the stimulated conditions I am using...
Here as some troubleshooting I've done so far:
1) same culture conditions. time, cell density, volume of incubation, time.
2) no mycoplasm contamination by PCR
3) no endogenous IFN-gamma cytokine in our medium nor FCS (checked by ELISA).
Any ideas?
Thank you in advance.
Alexis
Both LPS and bacterial DNA can induce IFN in NK cells. Sometimes this can come from autoclaved material.
NK cells can be stimulated by bacterial products like LPS, some virus and other bacterial cells to produce IFN. Please follow a published paper
The information is incomplete and in normal physiological conditions circulating IFN-gamma is a possible. In the NK92 culture the possible contaminants like LPS and DNA /RNA could possibly trigger IFN-gamma. Any possible DNA from lysed cells could possibly trigger the unexpected IFN-gamma peaking.
With your description so far, it is difficult to get any idea. It is known that some viruses cause such a response in some cultures. Hence, in case that you cannot exclude that, I would suggest that you start with a new lot of thawed cells from your frozen stock.
Hi all,
Thank you for your comments.
New batches of cells were sued as soon as we got this problem.
We even got cells form a different lab in case our hypothetical contamination was from an old time. but we are still getting that massive IFNg production.
I will have to check for DNA contamination or PAMPs leftovers after consumable sterilization.
Thank you
Hi Alexis,
We recently used this NK-92 cell line for a client at iQ Biosciences and noticed that the amount of IL-2 used in cultures to maintain the cells can impact the IFN-g secretion as well as passage number of the NK-92 line.
You may need to check if you have a new batch of IL-2 being used which isn't as "clean". We switched to the clinical grade of IL-2 in our cultures and have not had these problems since.
Some alternatives could be to use a different NK cell line such as the NKL to see if you are observing the same issues.
Good Luck,
Omar
Thank you for all of your answers.
For IL2 amount, we are currently using 100 U/mL and are changing the medium every 2 days.
I realized last week that the yellow tips we are using are not pyrogen free.
We will redo the experiment with pyrogen free tips to rule out this possible side-effect.
Alexis
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