Single-cell Ca2+ imaging was performed as previously described (8). In brief, freshly isolated T cells were loaded with Fluo4-AM (10 μM) and Fura Red-AM (20 μM) for 50 min at room temperature. After washing, the cells were resuspended in Ca2+ buffer [140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM Hepes (pH 7.4), 1 mM NaH2PO4, 5 mM glucose]. To stimulate the T cells, protein G beads (Merck Millipore) were coated with antibodies (anti-CD3/anti-CD28) according to the manufacturer’s instructions. Coverslips were coated with bovine serum albumin (5 mg/ml; Sigma-Aldrich) and poly-l-lysine (0.1 mg/ml; Sigma-Aldrich) to facilitate adherence of T cells. Imaging was carried out with a Leica IRBE2 microscope (100-fold magnification) using a Sutter DG-4 as a light source and an electron-multiplying charge-coupled device camera (C9100, Hamamatsu). Exposure time was 25 ms (40 frames/s) in 14-bit mode with twofold binning. A Dual-View module (Optical Insights, PerkinElmer Inc.) was used to split the emission wavelengths with the following filters: excitation (ex), 480/40; beam splitter (bs), 495; emission 1 (em1) 1, 542/50; em2, 650/57.

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