Fig. 5.

In vitro calibration of Fluo-4 fluorescence at varying pH. (A) In vitro calibration aimed to correlate Fluo-4 concentrations with Fluo-4 fluorescence at given calculated free [Ca²⁺] in a cuvette for vacuolar (5.2) and cytoplasmic (7.2) pH. For each bulk [Fluo-4], a sigmoidal curve was fitted to the data, yielding K_d values for the Fluo-4:Ca²⁺ binding. The expected free steady-state [Ca²⁺] region for the cytoplasm and DV is highlighted in grey. (B). Calculated average pooled K_d from the fitted curves to the normalized data in (A). The binding affinity is approximately two orders of magnitude lower at vacuolar over cytoplasmic pH (indicated by the greater K_d values at pH 5.2). (C) Using the K_d values from (B) and the fixed steady-state [Ca²⁺] in the respective compartments (Table 2), a [Fluo-4]-normalized fluorescence conversion curve was constructed to convert F/F(0) values from (Fig. 2) into absolute [Fluo-4].