T cells but not NK cells exhibit strong calcium signals in the presence of stimulatory DCs. (A) OT-I T cells or NK cells were cultured alone or with OVA peptide-pulsed DCs purified from poly I:C-treated mice (together or separated by a transwell). After 24 hours, CD69 up-regulation was measured by flow cytometry. (B-C) DCs were purified from poly I:C-treated transgenic mice expressing CFP and pulsed with the OVA peptide. OT-I CD8 T cells or NK cells were loaded with the calcium indicator Fluo3 and the SNARF dye and were either left alone or were incubated with DCs for 1 hour at 37°C. (B) T cells but not NK cells from stable conjugates with stimulatory DCs in vitro. The percentage of T cells (left panel) or of NK cells (right panel) conjugated to a DC is shown in red. (C) Flow cytometry was also used to analyze calcium signals in T cells or NK cells that were conjugated with a DCs at the time of data acquisition (red gate in panel B). The same analysis was performed with T cells or NK cells cultured in the absence of DCs (green gate). Histograms show the Fluo3 fluorescence in T cells or NK cells cultured alone (plain green histograms) or when conjugated to a stimulatory DC.