Metafluor software

GC cells cultured on coverslips were loaded with 5 μM Fura-2AM (Invitrogen, NY, USA) in physiological salt solution (PSS) at 22 °C for 50 min and then washed with PSS for 30 min. Then, cells on coverslips were placed in a standard perfusion chamber on the stage of an inverted fluorescence microscope (Nikon, Japan). The ratio of Fura-2 fluorescence with excitation at 340 or 380 nm (F340/380) was followed over time and captured with an intensified CCD camera (ICCD200). Images were acquired using a Meta Fluor software (Universal Imaging Corporation, Downingtown, PA). PSS used in digital Ca²⁺ measurement contained the following (in mM): 140 Na⁺, 5 K⁺, 2 Ca²⁺, 147 Cl⁻, 10 HEPES, and 10 glucose, pH 7.4. For the Ca²⁺-free solution, Ca²⁺ was omitted and 0.5 mM EGTA was added to prevent possible Ca²⁺ contamination.

The iodide-sensitive enhanced yellow fluorescent protein (EYFP-I152L) was used to measure anion conductance as described previously [18 (link)]. Cells grown on coverslips were continuously perfused at 4–5 mL/min with Ringer solution (37 °C). YFP-I152L-fluorescence was excited at 485 nm and CFP-fluorescence at 440 nm to identify TMEM16-CFP-CAAX-positive cells (Figure S1) using a high-speed polychromatic illumination system for microscopic fluorescence measurements (Visitron Systems, Puchheim, Germany). The emitted light at 535 ± 25 nm was detected with a Coolsnap HQ CCD camera (Roper Scientific). Quenching of YFP-I152L-fluorescence by iodide influx was induced by replacing 20 mM extracellular chloride by iodide. Images were analyzed with Metafluor software (Universal Imaging). Changes in fluorescence induced by iodide were expressed as initial rates of fluorescence decrease (ΔF/Δt).

TIRF illumination (TIRFi) was used for our experiments. The expanded beam of a 488/568 nm argon/krypton multiline laser (20 milliwatts, Laserphysics, Germany) passed through an AOTF laser wavelength selector (VisiTech International, UK) synchronized with a SNAP-HQ CCD camera (Roper Scientific, Germany) under Metafluor software (Universal Imaging, USA) control and was introduced to the coverslip from the high numerical aperture objective lens (Zeiss α-plan FLUAR 100X). Light entered the coverslip and underwent total internal reflection at the glass-cell interface. In our experimental conditions, penetration depth of TIRFi was calculated to be about 90 nm [17 (link), 25 (link)]. In single-wavelength TIRFi experiments (488 nm) the laser beam was filtered via the Zeiss filter set 10 and images were acquired at 20–40 Hz (Zeiss, Switzerland). In dual-wavelength TIRF illumination (488/568 nm), laser beams were combined by a dichroic mirror from the Zeiss filter 24 at 20–40 Hz. The pixel size was 126 nm (at binning 2).

Ratiometric single cell [Ca²⁺]_i imaging was performed on an IX-81 microscope (Olympus)-based system as described previously48 (link). HEK293 A cells and COS-7 cells were incubated in DMEM containing 2 μM Fura-2 AM at 37 °C for 45 minutes. PC-12 cells were incubated with 2 μM Fura-2 AM in Ca2 solution for 50 minutes. Data were acquired with Metafluor software (Universal Imaging) and analyzed with OriginPro 8 software (OriginLab) and are expressed as means ± S.E.

Intracellular Ca²⁺ variations ([Ca²⁺]_i) in cultured SMCs were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2 as previously described49 (link)50 (link). SMCs sub-cultured for 4 days in Lab-Tek II® chambers (Nunc, USA) were loaded with 2.5 μM Fura-2AM plus 0.02% Pluronic F-127. Cells rinsed with PSS were maintained in basal buffer during a 15-min waiting period for the de-esterification of Fura-2AM and chambers were mounted on a microscope stage (Axiovert, Zeiss, Germany; 20x objective). Buffer and drugs were then applied by perfusion to the cells as indicated in the figure legends. Cells were illuminated by excitation with a dual UV light source at 340 nm and 380 nm using a lambda DG-4 excitation system (Sutter Instrument Company, CA, USA). Images were captured digitally every 0.35 seconds with a cooled CCD camera (Photometrics, Roper scientific, France) at 510 nm emission. Changes in [Ca²⁺]_i were deduced from variations in the F340/F380 ratio after correction for background and dark currents (Metafluor software, Universal Imaging Corporation, USA). Data were averaged (at least 25 cells per field chosen randomly; one field per cover glass; 4 cover glasses for each experimental condition), with n representing the number of cell cultures.

The HC activity was evaluated using the Etd uptake method as described (Figueroa et al., 2013 (link)). In brief, sub-confluent HeLa cells grown on glass coverslips were washed twice with recording solution [in mM: NaCl (148); KCl (5); CaCl₂ (1.8); MgCl₂ (1); glucose (5); HEPES (5), pH = 7.4] containing 5 μM Etd. Basal fluorescence intensity from the nucleus of each cell was recorded for 5 min, using an Olympus BX 51W1I upright microscope (Olympus America Inc., Center Valley, PA, USA). Next, cells were washed three times with (Ca²⁺/Mg²⁺-free) DCFS and the fluorescence intensities of the nuclei were recorded. At the end of each experiment, the Cx HC blocker La³⁺ was added to confirm HC mediated Etd uptake (Contreras et al., 2002 (link); Retamal et al., 2007 (link)). Dye uptake of each cell was digitally photographed using a CCD monochrome camera (CFW-1310M; Scion; Frederick, MD, USA). Images were captured every 30 s (exposure time = 30 ms, gain = 0.5). Metafluor software (version 6.2R5, Universal Imaging Co., Downingtown, PA, USA) was used for data acquisition and off-line image analysis. The fluorescence intensity of at least 30 cells per experiment was averaged and plotted against time (expressed in minutes). Lastly, the slope, here called Etd uptake rate, was calculated using Microsoft Excel software and expressed as arbitrary units per minutes (AU/min).