I have tried different concentrations of LPS (1, 10 ug/mL), on THP-1 cells differentiated by PMA (1 x 10^6 cells/mL, or 2 x 10^6 cells/mL), at different time points (3h, 6h and 24h). There is no problem with the human IL-6 ELISA assay, as the IL-6 standards show a clear dose-dependent response. However, the activated cells (with LPS) had very low absorbance with the same incubation time with the standards.
The same samples and model was used to check TNF-a and it was successful.
Anyone can help with this situation? Thanks in advance!
Risking being brash I would say that the first thing I would check is whether the kit is for human IL-6, since THP-1 cells are of human origin.
Provided you are using the right kit, check my answer in the thread linked below.
https://www.researchgate.net/post/Why_LPS_didnt_induce_IL-6_and_IL-10_expression_in_THP-1_cells
I agree with Julie,
You may have better luck at later time points perhaps at 24-48h of incubation.
You may also consider increasing the total cell numbers in your reaction.
Such as 1X10e6 cells per 100ul, thereby increasing the overall concentration of the cytokines released in the medium.
Best
S
Hi there,
I have conducted a similar experiment previously in passing.
We took THP-1 monocytes differentiated by PMA for 48 hours.
We exposed them to 10 ng/mL E. coli LPS for 4 hours and left them to incubate for 24 hours in LPS-free media.
Using R&D Duoset Human IL-6 ELISA we found IL-6 in concentrations of 50-100 pg/ml.
Aside from the above advice you may optimise the incubation time with the colourmetric agent (TMB in our instance). Low expression of cytokines can benefit from a few extra minutes to develop colour.
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