Measuring Mitochondrial and ER Calcium

Measurements of [Ca²⁺]_mito and [Ca²⁺]_ER in HeLa single cells were performed as described previously^{51 (link),52 (link)}, using the genetically-encoded Ca²⁺ indicators CEPIA3mt (Addgene ID 58219) and G-CEPIA1er (Addgene ID 58215), respectively, which were developed by Dr. M. Iino (The University of Tokyo, Japan)^{53 (link)}. The constructs were introduced into HeLa cells utilising the X-tremeGENE HP DNA transfection reagent (Roche, Mannheim, Germany) according to the manufacturer’s protocol. The [Ca²⁺] measurements were performed 48 h after transfection using a Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20 × air objective and a high-speed digital camera (Axiocam Hsm, Zeiss, Jena, Germany). Changes in fluorescence were monitored in the GFP channel (Ex 480 nm, Em 520 nm). Extracellular Ca²⁺ was chelated with 3 mM EGTA, and PXA (10 μM) or thapsigargin (1 μM) were added as indicated on the figures. All traces were normalised (F/F₀) where F₀ is the starting fluorescence of each trace.