I want to do a DC:T cell co-culture and quantify the secretion of IL-17 and IFNg after culture. However the antigen specific cell population I will be using is rare (I expect to be able to recover ~1000-10,000 cells). Will this be enough cells to secrete a quantifiable amount of these cytokines as measured by ELISA? The T cells will already be primed in vivo, and this will be an in vitro/ex vivo restimulation.
Hi Josh,
I believe you should be able to see something in your supernates, if you keep the volume of supernatant down in your culture to a minimum to keep the cytokines concentrated - make sure your incubator is well-humidified so that you don't evaporate it all away! That said, it also depends on the sensitivity of your ELISA. I'd make sure to use reliable kits with good sensitivity that you've tested elsewhere.
I'll also note that qPCR is much more sensitive than ELISA for detecting upregulation of cytokines. Typically you can easily see cytokine mRNA upregulation even when you're well below the LOD of an ELISA on the protein side. If you're able to sample the cells and extract mRNA for qPCR, that would be my suggestion to make sure you can see any responses from your small numbers of cells.
Josh, have you considered ELISPOT instead of ELISA (or qPCR) as your experimental output? Please let me know if you have additional questions, I would be happy to help. Greg
Emily A Voigt and Greg A Kirchenbaum are both right. You could maximize the chances of picking up signals in ELISA by working with small media volumes and using high sensitivity kits. However, when working with low frequency T cell pools (usually the case with antigen specific t cells), ELISPOT is your best bet. This is a reasonably easy assay to set up and has really high sensitivity, which means you should be able to pick cells up that secrete either cytokine even when your responder frequency is super low. I notice that Greg A Kirchenbaum works at CTL. They are the experts in ELISPOT technology and so as he already mentioned, would be able to help with any Q you may have.
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