Dear colleagues,
we would like to perform a dual Fluorospot assay for evaluation of cytokine production (IL17A and IFNgamma) by tumour-experienced T cells.
What do you think would be best suited for the overnight stimulation of my cells, PMA + ionomycin, ConA or aCD3 + aCD28?
Many thanks
Tom
Tommaso,
PMA+ionomycin works fine. If you have opportunity (and funding) I'd suggest to try the BD FastImmune CD28/CD49d, which provides impressive results with fast stimulation.
Ricardo
I tried both CD3+CD28 and PMI+ Ionomicyin, both are fine. I would suggest to use PMI+Ionomycin and if you want to detect the cytokins in the cells with FACS for example you could add Brefeldin A to block vescicle exocitosis.
Dear Tommaso,
All the stimulants you mentioned will work, but for best suitable result, I will suggest you that after overnight result, if you add PMA 10 micromolar + Ionomycin 1 micromolar with Golgi mediated exocytosis blocker such as Brefeldin A or Monensin for 4 hrs before preparation of cells for assay.
Good luck
Just to mention that Brefeldin A or Monensin should be not used for assays when aiming the detection of cytokines in the supernatant (ELISPOT, Fluorospot, ELISAs, etc).
Another stimulus to consider is CD40. There is accumulating evidence that this is a costimulatory molecule on T cells which can lead to production of both IFNg and IL-17. Try CD40 alone as well as CD3+CD40 and CD3+CD40+CD28. Generally anti-CD40 clone 1C10 generates the best response but it may be necessary to try other ab's.
Good Luck!
Gisela
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