Fura-2-am

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 experiments using Fura-2, unless otherwise noted, Imaging Buffer was Ca²⁺ containing Hanks Balanced Salt Solution (Ca²⁺:HBSS) (Gibco 14025–092) containing any relevant drugs (i.e. PLX4720). To cells in 35 mm dishes, 1 mL of medium was removed and Fura-2-AM was added to 4 μM (2X) concentration before adding back to the plate to achieve a 2 μM final concentration. Plates were incubated at 37°C, 5% CO₂ for 30 minutes. After dye loading, the media was aspirated and the plates were washed 3X with 2 mL of Imaging Buffer warmed to 37°C. After the final wash, 2 mL of Imaging Buffer was added to the dish.

After dye loading/washing, imaging dishes were moved to a heated stage. Fura-2-AM fluorescence (Ratio 340Ex/380Ex-535Em; 340/380 ratio) was measured every 5 seconds as an indicator of intracellular Ca²⁺; when the intracellular concentration of Ca²⁺ increases, so does the 340/380 ratio. Imaging was performed for a period of 40 minutes with a Nikon Eclipse Ti2 microscope using a 10X objective and equipped with a Photometrics Prime 95B 25mm sCMOS Camera. For Ca²⁺ free conditions, cells were washed and imaged in Ca²⁺ free HBSS (Gibco 14175–095).

SOCE assays were conducted, as described previously⁸⁷ , in three phases consisting of 1) an equilibration period in Ca²⁺-free HBSS, 2) release of Ca²⁺ from the endoplasmic reticulum (ER), and 3) the induction of SOCE activity. Changes in cytoplasmic Ca²⁺ levels were measured with Fura-2-AM, as described above. Briefly, cells were dye loaded, washed, and incubated in Ca²⁺ free HBSS before imaging. Phase 1) Ca²⁺-free HBSS was perfused over cells for 10 minutes of imaging to allow equilibration and to acquire a baseline. Phase 2) Ca²⁺-free HBSS buffer containing 50 μM of the sarco-endoplasmic Ca²⁺ ATPase (SERCA) inhibitor, cyclopiazonic acid (CPA), was perfused over cells for 8 minutes. During this phase, CPA treatment leads to the release of free Ca²⁺ within the endoplasmic reticulum (ER), allowing for the quantification of ER Ca²⁺ stores and subsequent cytoplasmic Ca²⁺ clearance. Release of ER Ca²⁺ stores results in activation of SOCE channels, however, they do not contribute to the Ca²⁺ signal until extracellular Ca²⁺ is added to the assay in the third phase. Phase 3) Perfusion with Ca²⁺:HBSS (+50 μM CPA) allows influx of Ca²⁺ through activated SOCE channels, and subsequent quantification of SOCE activity on a cell by cell basis. This final component of the assay proceeds for an additional eight minutes before imaging is stopped. For all phases, cells were perfused at a flow rate of 2 mL/min. Quantifications of ER Ca²⁺ content and SOCE activity are calculated by taking the integral of each cell trace within the respective phases, followed by baseline subtraction.