Coolsnap hq

Renal primary cells were loaded with 2 µM Fura-2/AM and 0.02% Pluronic F-127 (Invitrogen, Darmstadt, Germany)) in ringer solution (mmol/l: NaCl 145; KH₂PO₄ 0.4; K₂HPO₄ 1.6; Glucose 5; MgCl₂ 1; Ca²⁺-Gluconat 1.3) for 1 h at room temperature. Fluorescence was detected in cells perfused with Ringer’s solution at 37 °C using an inverted microscope (Axiovert S100, Zeiss, Germany) and a high speed polychromator system (VisiChrome, Puchheim, Germany). Fura-2 was excited at 340/380 nm, and emission was recorded between 470 and 550 nm using a CoolSnap camera (CoolSnap HQ, Visitron). [Ca²⁺]_i was calculated from the 340/380 nm fluorescence ratio after background subtraction. The formula used to calculate [Ca²⁺]_i was [Ca²⁺]_i = Kd × (R − R_min)/(R_max− R) × (S_f2/S_b2), where R is the observed fluorescence ratio. The values R_max and R_min (maximum and minimum ratios) and the constant S_f2/S_b2 (fluorescence of free and Ca²⁺-bound Fura-2 at 380 nm) were calculated using 1 µmol/l ionomycin (Calbiochem), 5 µmol/l nigericin, 10 µmol/l monensin (Sigma), and 5 mmol/l EGTA to equilibrate intracellular and extracellular Ca²⁺ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2•Ca²⁺ complex was taken as 224 nmol/l.

The plasma membrane bound calcium sensor GCaMP6 was fused to the N-terminus of CFTR. HEK293 cells grown on coated glass cover slips were transfected with GCaMP6-CFTR, and were mounted in a perfusion chamber 72 hrs after transfection. Cells were perfused with ringer solution at a rate of 8 ml/min at 37 °C. Cell fluorescence was measured continuously with an inverted microscope Axiovert S100 (Zeiss) using a x40 objective (Fluar 40x/1.3 Oil, Zeiss) and a high speed polychromator system (VisiChrome, Visitron, Puchheim, Germany). GCaMP6-CFTR was excited at 485 nm and 405 nm. Emission was recorded between 520 and 550 nm using a CCD-camera (CoolSnap HQ, Visitron). Control of experiments, imaging acquisition, and data analysis were done with the software package Meta-Fluor (Universal imaging, New York, USA). Alternatively, cells were loaded with Fura2 and intracellular Ca²⁺ concentrations were determined as described earlier^{49 (link)}.

Measurement of the global cytosolic Ca²⁺ changes were performed as described recently [41 (link)]. In brief, cells were loaded with 5 µM Fura-2, AM (Molecular Probes) in OptiMEM (Invitogen) with 0.02% pluronic (Molecular Probes) for 1 h at RT and 30 min at 37 °C. Fura-2 was excited at 340/380 nm, and the emission was recorded between 470 and 550 nm using a CCD-camera (CoolSnap HQ, Visitron Systems, Germany). Control of experiment, imaging acquisition and data analysis were done with the software package Meta-Fluor (Universal imaging, USA). [Ca²⁺]_i was calculated from the 340/380 nm fluorescence ratio after background subtraction. The formula used to calculate [Ca²⁺]_i was [Ca²⁺]_i = Kd × (R − R_min)/(R_max − R) × (S_f2/S_b2), where R is the observed fluorescence ratio. The values R_max and R_min (maximum and minimum ratios) and the constant S_f2/S_b2 (fluorescence of free and Ca²⁺-bound Fura-2 at 380 nm) were calculated using 1 µmol/liter ionomycin (Calbiochem), 5 µmol/liter nigericin, 10 µmol/liter monensin (Sigma) and 5 mmol/liter EGTA to equilibrate intracellular and extracellular Ca²⁺ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2•Ca²⁺ complex was taken as 224 nmol/L.