I'm trying to optimise an ICS protocol for assessing cytokine production in human PBMCs. While I have good staining for IFN-g and TNF-a using either PMA/ionomycin or CD3/CD28 stimulation, I am unable to get any signal for IL-10 and only non-specific (unstimulated same as stimulated) signal for IL-4. I have tried two different antibodies for each cytokine, as well as monensin vs brefeldin. I have tried short incubations (6 hours) as well as a 24 hour incubation.
Any suggestions would be much appreciated!
Thanks.
IL4 production is restricted to Th2 cells whereas IL10 is produced by activated CD8 and CD4 Tcells. Since you use Tcell specific activations Bcell and monocyte contribution would be much less. I recommend using ELISA in parallel to be able to quantify secreted proteins as well. You can use Burkitt Lymphoma line as positive control for IL10 assay.
IL4 production is restricted to Th2 cells whereas IL10 is produced by activated CD8 and CD4 Tcells. Since you use Tcell specific activations Bcell and monocyte contribution would be much less. I recommend using ELISA in parallel to be able to quantify secreted proteins as well. You can use Burkitt Lymphoma line as positive control for IL10 assay.
This is a common issue in mouse immunology as well. Thus the IL-4 and IL-10 reporter mice have been created which express a congenic cell surface marker when the gene's promoter is activated. (This however will not help you with human cells).
I addition for Korcan's comment, you could also try to enumerate the number of cells secreting these cytokines via an Elispot assay. Another possibility is to use a bispecific (cell surface) technique which is similar to an Elispot but compatible with flow cytometry. (https://www.miltenyibiotec.com/~/media/Images/Products/Import/0001200/IM0001207.ashx) It is an interesting twist on their Mac's technology.
Thanks Korcan and David. I had planned to look into Luminex or Elisa to measure secreted cytokines, but the MACS technique is also very interesting.
Which antibodies use? I detected signal of IL-10 using ebioscience antibody in lymphocytes. For IL-4, we detected intracellular production using BD antibody in lymphocytes.
We observed the best signalling of IL-10 using a CD3/CD28/CD2 stimulation for 48h, and prior to analysis 5h of culture with monensin.
In monocytes, LPS culture for 12h in presence of monensin is sufficient to detect IL-10 production
I'm using the BD Cytofix/Cytoperm kit. Using a BD PE conjugated IL-10 antibody as well as Biolegend BV421 conjugated IL-10 antibody.
Are you stimulating whole PBMCs with CD3/CD28/CD2? What proportion of CD4 cells are making IL-10?
Thanks.
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