Hi,
I prepared cell culture from mice lymph nodes. I did ELISA for IL6; however, the levels of IL6 were really low to detect at 48hrs, 72hrs, and 96hrs.
I have read in the literature that to detect IL-6 using ELISA is more suitable at 6hrs and 24hrs. Any suggestions?
You should try at 24 h. This is the peak time point for cytokine expression.
Hi Mishaal,
This can vary depending on the cell type etc, but as a general rule you need to give your cells enough time to secrete a detectable amount of protein.
When I measure IL6 I wait 24hrs, but if your cells also use up the IL-6 in culture the change may be difficult for you to detect whatever time point you use.
If you have the materials I would recommend a preliminary testing several time points first, there is no point going straight in before you optimise the experiment. 6 hours is the lowest time point I would personally use.
As Rachel mentioned, 6H would be the shortest time point I would use. 24H is much better as most cytokine levels (except for TNF) go up with time. Provided your cells did secrete IL-6, you should have been able to pick IL-6 up at later time points that you tested, as long as your cells didn't use up the secreted IL-6.
One other consideration would be the number of cells and the volume of media that you plated the cells in. Are the cells numbers high enough for the amount of media? IL-6 is a cytokine that is secreted at super high levels by splenocytes and BMM and we used to plate 10e6 splenocytes or 3x10e5 BMM in 300 microL to get good signals for most cytokines (IL-6 would be through the roof). Maybe you need to plate a lot more of your cells in a smaller volume. Just a thought.
Also what are you stimulating the cells with? If you add a positive control and see IL-6 production there but not with your stimulant, then you might have to fiddle with your stimulant concentrations as well.
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