Hello,

I would not use anti-CD3/CD28 stimulation in an antigen-specific assay: it will probably induce polyclonal activation of T-cells (and not only the Ag-specific ones...) and increase your background production of cytokines in the negative control well, rendering possibly impossible to identify a Ag-specific stimulation in Ag wells. In our hands, anti-CD28/CD49d induces minimal background (at least in 6h or overnight stimulation). Hope it helps.

The choice between anti-CD3/CD28 and anti-CD28/CD49d depends on your experimental goal. Anti-CD3/CD28 activates the TCR via CD3 and provides co-stimulation via CD28, inducing both proliferation and cytokine secretion, making it essential for antigen-specific responses. In contrast, anti-CD28/CD49d primarily amplifies cytokine secretion without significant TCR activation and is more suited for general cytokine studies. Using all three (CD3, CD28, and CD49d) is possible but may result in excessive activation, potentially masking antigen-specific effects. Additionally, note that Tregs do not express CD49d, meaning anti-CD28/CD49d will not activate them, whereas anti-CD3/CD28 could. Pilot experiments can help determine the best approach for your system.