Single-cell ER Ca2+ Measurement Protocol

Single-cell ER Ca²⁺ measurements were performed as previously described^{39 (link)}. ER Ca²⁺ levels were measured with the genetically encoded Ca²⁺ indicator G-CEPIA1er, which was kindly provided by Dr. M. Iino (The University of Tokyo, Tokyo, Japan)²⁶ . The G-CEPIA1er construct was introduced into HeLa cells utilizing X-tremeGENE HP DNA transfection reagent (Roche, Mannheim, Germany, 06366546001) according to the manufacturer’s protocol. A Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20× air objective and a high-speed digital camera (Axiocam Hsm, Zeiss, Jena, Germany) were used for these measurements. Changes in fluorescence were monitored in the GFP channel (480/520 nm excitation/emission). To chelate extracellular Ca²⁺, EGTA was added to a final concentration of 3 mM. One minute later, 1 µM TG or different SOCE inhibitors were added. All traces were normalized (F/F₀) where F₀ is the starting fluorescence of each trace.