Analyses of fura-2 Ca²⁺ binding affinity in situ. (A) Comparison of the viscosity changes according to the concentration of protein (aldolase) or sucrose. From a linear regression of the measured value, the viscosity of a 20-mg/ml protein-containing solution was equal to that of a 283-mM sucrose-containing solution (dotted line, n = 9). (B) Cartoon showing the condition for the in vitro calibration of fura-2 in the thin layer of solution between the two coverslips. (C) In vitro calibration (fluorescence [F₃₈₀]–pCa relation) of salt (control), sucrose-containing solution, and protein-containing solution using EGTA-buffered Ca²⁺ calibration solutions. The fluorescence–pCa relation was shifted to the right by 283 mM of sucrose and 20 mg/ml of protein (n = 8, 8, and 12 for control, sucrose, and protein, respectively). (D) Summary of the K_d,fura obtained from the fluorescence–pCa relation.