Hi Priya

Do you know for sure that the cells secrete the tested cytokines? Sometimes the cells do not secrete them (might still express the protein within the cells, but unable to secrete it in the medium). In that case you would not get the ELISA to detect anything. If you are not confidant whether they do in fact secrete the cytokines, maybe you can test the desired cytokines with the cell lysates (instead of using the extracellular media).

Also, I am assuming that you had a positive control in your experiments, and your positive control showed the presence of desired cytokines..?

Best,

Tasfia

Thank you very much for the input. I feel the cell lysate will help in getting a clearer picture.

You haven't specified which cells you're using and at what density, and how long you are leaving them for. One of the most likely things is that you simply don't have enough cells/not leaving them long enough to build up sufficient levels of the cytokines in the medium to detect.

 Hi! I am using Mesenchymal stem Cells in 80% confluent T75 flask, treated with LPS for 24 h.

At what concentration are using LPS? What is the source of your LPS?

You should stabilize your LPS with 5% human albumin.

Thanks LPS is from bacterial source. Shall keep note of stabilizing LPS with albumin. Can you please share the protocol of stabilizing the LPS. 

5% human albumin is a drug in clinical medicine. Just add your LPS to this solution and freeze aliquots of it at -30°C. For thawing room temperature is ok.

Hello Priya,

I used ELISAs to confirm RT-qPCR results for some inflammatory cytokines in mice epithelial and endothelial cell lines, after stimulation with cyclosporin and tacrolimus and used LPS as a positive control for this responses, and got good results from supernatants but with several caveats:

1) What Ms. Rakib has said. 

2) Make sure you use the appropriate ELISA (I used against MCP-1 and IL-6 and, also, as endothelial activation is an event concurrent with inflammation, ICAM-1) and that the expression kinetics are optimal: for instance, the MCP-1 response was acute and I got very evident levels of this cytokine 3 hours and lower, but more stable, at 6 hours but by 24 hours they were even lower. With ICAM-1, on the other hand, 24 hours was optimal.

3) The gene expression levels through RT-qPCR were not a good starting point to extrapolate on the direct protein detected in the supernatants, neither for quantity nor for time. You may want to test different times and don't let the relative gene expression levels set an expectation for how high should the direct expression be.

4) Obvious things (which is where most times everything fails): conserve your samples at -80ºC, prepare an LPS calibration curve every time, make sure your buffers have the correct pHs for your ELISA and if you have the slightest doubt make them anew, and, in case you do it, do not dilute your supernatant samples before loading them in the plate or reduce their dilution (good ELISAs are very sensitive but sometimes the samples may fall out of range if you dilute them).

Hope some of this helps.

Thanks for the input Carlos. I am looking for interleukins at longer time periods starting from 24h. Alongside also planning qPCR. In your qPCR did u also check  for gene expression of MCP1 and ICAM 1, for 3h and 6h after treatment with LPS?