You may have conduct a kinetic study including 6 12 and 24 h post-treatment. It would be helpful to also validate it on isolated PBMCs using ELISA.  

Dear Olwyn,

The ELISA idea is a good one, but another thing to bear in mind is interdonor variability as far as these responses are concerned. Given the diverse make up of PBMC populations in different individuals, it is very possible to get equally diverse cytokine responses. Perhaps, repeat this assay over a range of donors (n= 3-5?). It's a pain, of course, to isolate/purify the cells, stimulate and test, but it may all be worth it in the end! 

All the luck in the world!

A. 

Dear Olwyn,

I beg to differ on the above comments made by my esteemed colleagues. Intracellular cytokine and measured cytokine in culture supernatant usually do not correlate. We recently published a paper where we addressed some of these issues (one of the reviewers actually had asked us to show if they correlate). Please have a look.

Non-Classical monocytes display inflammatory features: Validation in Sepsis and Systemic Lupus Erythematous Scientific Reports 5, Article number: 13886 (2015)

doi:10.1038/srep13886

Now coming to the problems you are facing. There could be multiple reasons:

1. When you say lower levels than what is reported in literature, it could be that they used a different clone of the antibody. Did you check that?

2. I agree with Prof. Dileepan that you may have to do a time kinetics study. My recommendation would be to start at 2 hours and increase in 2 hour increments thereafter. 

3. What cells are you gating on? In other words, which cells (B cells, T cells, Monocytes, etc. I am assuming it's T cells) are you checking in terms of IFN-y production. There usually is cell to cell variability in cytokine production.

4. How many hours are you incubating with BFA? I usually don't incubate with BFA for more than 2 hours (for whole blood stimulation). Prolonged incubation with BFA can be toxic to cells which could vitiate your readout. 

5. What is the fluorochrome conjugate with your antibody? You can try using brighter dyes with high stain index, e.g. PE, APC/Alexa 647, or the Brilliant Violet series of dyes (assuming you have a violet lase in your flow cytometer).

I hope I did not confuse you too much. You can get in touch with me if you have doubts.

Good luck

Ratan