I want to detect the release of IL-6 and TNF-alpha upon LPS stimulation in RAW 264.7 cell line using ELISA. I have done the experiments previously where i did see the desirable results. But now, there is no release of IL-6 that can be detected upon LPS stimulation whereas TNF-alpha is being released.
I use 20ng/ml of LPS for stimulation but in incomplete media.I have tried using the fresh lot of LPS, increased the cell density and even increased the LPS concentration to 100ng/ml but IL-6 release cannot be detected but TNF-alpha release is seen.
What time point are you collected supernatants? from experience with raw cells, they secrete very little IL-6. Perhaps try a different ELISA kit.
Hi Ben,
I have collected the supernatant after 24 hrs of LPS stimulation.
I don't think there is any problem with the kit as standards are working quite well.
Perhaps you should try collecting supernatant after 4 hours or so, i have experienced a sharp increase in IL6 and TNFa at 4 hours that is significantly higher than at 24 hours in raw264.7. Whilst TNFa increases to several hundred pg/ml, IL6 typically reaches sub 100pg/ml
I am facing somewhat a similar situation where I cannot see the same kind of changes in morphology of my cells when stimulated with LPS, which I observed during earlier experiments. Also, similarly with the IL-6 levels.
The only variation is the plate type, earlier I used 6 wells plate now I am using 24 wells plate.
What worked for you? Gagandeep Kaur
Same here, I treated my RAW with 1 micro gram /mL LPS and cultured 24 h for ELISA Assay.
I have seen a dramatic increase in 3 h stimulated LPS sample qPCR
but havnt seen dramatic increase in TNF-alpha and IL1 beta in ELISA
thanks! i think i should go over 4h treatment for supernatant collection
For detecting different cytokines you need to do kinetics….. they increase at different time points!
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