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For calcium-flux experiments, CD8+ T live cells were loaded with Fluo-4AM (Molecular Probes, F10489) at 5 μM for 60 min in the presence of 2.5 mM probenecid. T cells were transferred to imaging medium (RPMI without phenol red, 10% FBS, 25 mM HEPES) and allowed to adhere to the non-stimulating coverslips. Engagement with live T2 cell previously loaded with a cognate peptide, lead to T cell activation. We quantified Fluo-4 responses by determining the average intensity of a region within each cell as a function of time using the ImageJ program (NIH).
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