Extended Data Figure 2. Calibration of fluorescence measurements with Ca²⁺ indicators.

a. Fluo-4 Ca²⁺ titration curve. Shown is the fraction of Ca²⁺-bound Fluo-4 bound at the indicated added [Ca²⁺]. Each point is a triplicate average. Titration curves are carried out in the indicated buffers. b. Fluo-4FF Ca²⁺ titration curve. Shown is the fraction of Ca²⁺-bound Fluo-4FF at the indicated added [Ca²⁺]. Each point is a triplicate average. c. Rhod-2 Ca²⁺ titration curve. Shown is the fraction of Ca²⁺-bound Rhod-2 at the indicated added [Ca²⁺]. Each point is a triplicate average. Titration is done in the absence and presence of PVP (polyvinylpyrrolidone) in the assay buffer. d. Ca²⁺ titration curve for Fura-2AM- or Rhod-2AM-loaded mitochondria. Shown is the fraction of Ca²⁺-bound Fura-2 or Rhod-2 at the indicated free Ca²⁺ concentration in the mitochondrial matrix ([Ca²⁺]_m). Each point is a triplicate average. 1 μM FCCP and 1 μM of the Ca²⁺ ionophore ionomycin are used to equilibrate [Ca²⁺]_m with the extra-mitochondrial free [Ca²⁺] (i.e., [Ca²⁺]_{extra, free})_. The [Ca²⁺]_{extra, free} is measured with Fluo-5N. Equations fit to the data for panels a-d are detailed in the main methods section.