Fig. 2.

Fluorescence from leaked Mag-Fluo-4 is quenched by QAb. (A) Permeabilized HEK cells loaded with Mag-Fluo-4 AM were re-suspended in CLM-H (i.e. CLM supplemented with HEDTA to provide effective buffering of high free [Ca²⁺]), with the indicated [Ca²⁺]_m and CPA (10 μM). We note that with the prolonged incubations used (5-20 min), luminal [Ca²⁺] and [Ca²⁺]_m equilibrate without the need for ionomcyin to accelerate equilibration (see Fig. 3A). Results (means ± SEM, n = 3 independent experiments each with duplicate determinations) show parallel analyses of the medium alone (after removal of cells by centrifugation, F_medium) or cells in the medium (F_{permeabilized cells}). (B) Results from panel A were used to estimate the contribution of leaked indicator to total fluorescence intensity (F_medium/F_{permeabilized cells,} %) at each [Ca²⁺]_m. (C) Fluorescence recorded from permeabilized cells after treatment with probenecid (2.5 mM). Means ± SD from a single experiment, typical of two experiments, each with duplicate determinations. (D) Effects of QAb (1:20) on Ca²⁺-dependent changes in fluorescence from leaked Mag-Fluo-4 (F_medium). Means ± SEM, n = 3 independent experiments, each with duplicate determinations (control trace reproduced from panel A). (E) Comparison of the responses of trapped Mag-Fluo-4 (F_{permeabilized cells} - F_medium) to changes in [Ca²⁺]_m in the absence or presence of QAb (1:20). Means ± SEM, n = 3 independent experiments, each with duplicate determinations. (F) Effects of QAb (1:20) on Ca²⁺-dependent changes in fluorescence from permeabilized cells together with their medium (F_{permeabilized cells}), or from trapped indicator alone (F_{permeabilized cells} - F_medium) in the absence of QAb. Means ± SEM, n = 3 independent experiments each with duplicate determinations (blue symbols reproduced from panel E). The results confirm that most fluorescence from leaked indicator is quenched by QAb. (G) Ca²⁺ uptake by permeabilized cells loaded with Mag-Fluo-4 AM and incubated in CLM with free [Ca²⁺]_m = 300 nM was stimulated by addition of ATP (1.5 mM). QAb (1:20) had no effect on the response. (H) Concentration-dependent effects of IP₃ on Ca²⁺ release from permeabilized HEK cells in CLM with 300 nM [Ca²⁺]_m. QAb (1:20) had no effect. To minimize use of costly QAb, results in G and H (means ± SD) are from a single experiment with 3 replicates.