Ionomycin

Cultured cells were transfected using Lipofectamine 2000 (Invitrogen) 2 or 3 days before imaging. Jurkat T cells were electroporated using a MicroPorator (MP-100, Digital Bio) 1 day before imaging. For cytosolic Ca²⁺ imaging using fura-2, cells were loaded with 5 μM fura-2 AM (Molecular Probes, USA) at room temperature (22–24 °C) for 40–60 min in 0.1% BSA-supplemented physiological salt solution (PSS) containing (in mM) 150 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 5.6 glucose and 25 HEPES (pH 7.4). Before imaging, the loading solution was replaced with PSS without BSA.
The images were captured using an inverted microscope (IX81, Olympus, Japan) equipped with a × 20 objective (numerical aperture (NA)=0.75, UPlanSApo, Olympus) or a × 40 objective (NA 0.90, UApo/340, Olympus), an electron-multiplying cooled-coupled device (EM-CCD) camera (ImagEM, Hamamatsu Photonics, Japan), a filter wheel (Lambda 10-3, Sutter Instrument, USA), a xenon lamp (ebx75) and a metal halide lamp (EL6000, Leica, Germany) at a rate of one frame per 2 or 3 s with the following excitation/emission filter settings: 472±15 nm/520±17.5 nm for G-GECO1.1, CEPIA1er, G-CEPIA1er, CEPIA2–4mt and EYFP-er; 562±20 nm/641±37.5 nm for R-GECO1, R-CEPIA1er and mCherry-STIM1; 377±25 nm/466±20 nm and 377±25 nm/520±17.5 nm for GEM-GECO1 and GEM-CEPIA1er; 340±13 nm/510±42 nm and 365±6 nm/510±42 nm for fura-2; 440±10.5 nm/480±15 nm and 440±10.5 nm/535±13 nm for D1ER19 (link)20 (link). For analysis of the ratiometric indicators, we calculated the fluorescence ratio (F₄₆₆/F₅₂₀ for GEM-GECO1 and GEM-CEPIA1er; F₃₄₀/F₃₆₅ for fura-2; F₅₃₅/F₄₈₀ for D1ER). Photobleaching was corrected for using a linear fit to the fluorescence intensity change before agonist stimulation. All images were analysed with ImageJ software.
To image subcellular ER Ca²⁺ dynamics during agonist-induced Ca²⁺ wave formation, we imaged HeLa cells expressing either G-CEPIA1er or R-CEPIA1er. Images were captured at a rate of one frame per 30–100 ms using a × 60 objective (NA 1.45, PlanApo TIRF, Olympus) and the metal halide lamp or an LED lamp (pE-100, CoolLED, UK). To evaluate Ca²⁺ wave velocity in the ER and cytosol, images were normalized by the resting intensity, and a linear region of interest (ROI) was defined along the direction of wave propagation. A line-scan image was created by averaging 30 adjacent linear ROIs parallel to the original ROI, and time derivative was obtained to detect the time point that showed maximal change during the scan duration. Then, the time points were plotted against the pixel, and the wave velocity was estimated by the slope of the least-squares regression line.
For mitochondrial Ca²⁺ imaging with ER and cytosolic Ca²⁺, mitochondrial inner membrane potential or mitochondrial pH at subcellular resolution, we imaged HeLa cells with a confocal microscope (TCS SP8, Leica) equipped with a × 63 objective (NA 1.40, HC PL APO, Leica) at a rate of one frame per 2 or 3 s with the following excitation/emission spectra: R-GECO1mt (552 nm/560–800nm), G-CEPIA1er (488 nm/500–550 nm) and GEM-GECO1 (405 nm/500–550 nm); GEM-GECO1mt (405 nm/500–550 nm), JC-1 (488 nm/500–550 nm and 488 nm/560–800nm); R-GECO1mt (552 nm/560–800nm), SypHer-dmito (405 nm/500–550 nm and 488 nm/500–550 nm). For analysis of JC-1 and SypHer-dmito, we calculated the fluorescence ratio (488 nm/560–800 nm over 488 nm/500–550 nm for JC-1 (ref. 55 (link)); 488 nm/500–550 nm over 405 nm/500–550 nm for SypHer-dmito62 (link)).
To perform in situ Ca²⁺ titration of CEPIA, we permeabilized the plasma membrane of HeLa cells with 150 μM β-escin (Nacalai Tesque, Japan) in a solution containing (in mM) 140 KCl, 10 NaCl, 1 MgCl₂ and 20 HEPES (pH 7.2). After 4 min treatment with β-escin, we applied various Ca²⁺ concentrations in the presence of 3 μM ionomycin and 3 μM thapsigargin, and estimated the maximum and minimum fluorescent intensity (R_max and R_min), dynamic range (R_max/R_min), K_d and n.
For the estimation of [Ca²⁺]_ER based on the ratiometric measurement using GEM-CEPIA1er (Figs 1e,f and 5b and Supplementary Fig. 5f), [Ca²⁺]_ER was obtained by the following equation:

where R=(F at 466 nm)/(F at 510 nm), n=1.37 and K_d=558 μM.
To evaluate pH-dependent change of EYFP-er fluorescence (Supplementary Fig. 4a–d), we stimulated HeLa cells expressing EYFP-er in a PSS (adjusted to pH 6.8) containing monensin (10 μM, Wako) and nigericin (10 μM, Wako). Subsequently, the cells were alkalinized with a solution containing (in mM) 120 NaCl, 30 NH₄Cl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 5 HEPES and 5.6 Glucose (pH 7.4)67 (link).

For in vitro T cell stimulation assays, PMA (Sigma-Aldrich) was used at 50 ng/ml and ionomycin (Sigma-Aldrich) was used at 1 μg/ml final concentrations. For CsA (Calbiochem) treatment, 10x stock solutions were prepared in EtOH. CsA stocks were diluted with olive oil (Sigma-Aldrich) and, after thorough mixing, were injected i.p. at a dose of 80 mg/kg. For tacrolimus (Calbiochem), the final treatment dose was 2.5 mg/kg.

Cell preparations were suspended in 1 ml loading buffer (DPBS supplemented with 1 mM CaCl₂, 1 mM MgSO₄, and 1% FBS) at a density of 1 × 10⁷ cells per ml and loaded with the calcium indicator dyes Fluo-4 (Thermo Fisher Scientific) and FuraRed (Thermo Fisher Scientific) at 37°C according to manufacturer’s directions. Cells were then washed and stained for flow cytometry at room temperature. Immediately before measurement, cells were incubated at 37°C for 5 min and then transferred to the flow cytometer for continuous monitoring. After obtaining a baseline measurement for 30 s, anti-CD3 antibodies (5 μg/ml) and IgG crosslinking antibodies (25 μg/ml) were added to the sample. After 180 s, ionomycin (1 μg/ml) was added to stimulate maximal cytosolic calcium influx.