I am planning to detect the surface expression of ICOS from PBMC. I need to know an exact methodology to activate T cells invitro to stimulate ICOS expression. Please assist.
I have stimulated PBMC with CD3/CD28 beads at bead: cell ratio of 1:10 for 72 hours and have seen >70% ICOs expression. In addition you could also culture your PBMC with Tetanus toxiod at 1:100- 1:1000 for 3-5 days for ICOS expression
3 days stimulation with PHA (5-10ug/ml) increases ICOS up to 60-70% on T cells, depending on the donor.
Thanks. These patients are suspected to have CVID. Do you think, Just PHA would activate and stimulate PBMCs to express ICOS or do I have to have a cytokine cocktail along with PHA? Do you have a published protocol which I can use?
I have stimulated PBMC with CD3/CD28 beads at bead: cell ratio of 1:10 for 72 hours and have seen >70% ICOs expression. In addition you could also culture your PBMC with Tetanus toxiod at 1:100- 1:1000 for 3-5 days for ICOS expression
Thanks Dr Padmalal. I would probably go with the CD3/28.There are functional grade CD3 and CD28.2 mabs which might give good results as beads. Do you have any previous experiences with these mabs? How about IL-2?
Iused CD3/CD28 magnetic beads from Miltenyi biotech and do not have any experince with IL-2 for this assay
Hi, usually with aCD3/CD28 Dynabeads you get strong ICOS expression... CVID patients may have defects in TCR signaling, so I would try : 1) purified CD4 with aCD3/CD28 Dynabeads + IL-2 on day2 post-stimulation and 2) total PBMC with PHA and IL-2. With this, you can do a time course of ICOS expression. Good luck
Thank you Dr Sara, Appreciate your contribution. How good would it be, if we use CD3/CD28.2 mabs (eBioscience) along with IL-2 instead of magnetic beads or PHA?
it's ok (anti-cd3 immobilized, anti-cd28 soluble in this case) but to be honest the beads, even if expensive, are much better stimulator. However, one problem with the beads is that they have a fixed concentration of Ab and you can't change that, while if you do the coating on the plate you can titrate the dose of anti-CD3, which I would advise to do.
I much appreciate your suggestions. Yes Anti-CD3 for initial coating and anti-CD28 for the subsequent activation signals. True beads are quite expensive and it's out of my budget. So I'll stick in to the mAbs and IL-2 if required. Thanks again for your help
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