P3000mp

Co-cultured astrocytes and Müller cells were loaded for 45 min at 37°C under gentle agitation with 2, 5 µM of Fura-2 acetoxymethylester (Fura-2AM, Molecular Probes, United States) in a modified Hanks buffered saline solution (HBSS: 135 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 2.5 D-glucose, and pH 7.4) supplemented with 0.002% pluronic acid (P3000MP, Molecular Probes, United States). After incubation, glass plates covered with cells were mounted in a thermostatically regulated microscope chamber on an inverted Olympus microscope (IX 70, Olympus Corporation, Japan) and visualized with a 20X objective. Intercellular calcium waves were induced by the application of 100 µM ATP onto a single Müller cell with an 8.4-µm glass micropipette. Two 100 msec-pulses at 10 psi were delivered with a pneumatic PicoPump (PV830, World Precision Instruments, United States). A real-time movie of intercellular calcium waves of stimulated Müller cell and neighboring astrocytes following stimulation was recorded at 2 Hz for 4 min by alternating excitations at 340 and 380 nm (emission spectrum: 420–600 nm). Images were recorded using a CCD camera with the live acquisition software (TiLL photonics, United States). The 340/380 nm fluorescence ratio was calculated after correction for background fluorescence values. A baseline recording was performed for 2 min before stimulation.

A549 cells were attached onto coverslips and loaded with 6 μM 2′,7′-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM, 0061, TEFlabs) along with the same volume of 0.05% pluronic acid (P-3000MP, Invitrogen) for 15 min at room temperature (RT) in the dark. After incubation with the BCECF, the A549 cells were placed on the inverted microscope and perfused with physiological salt solution, as previously described (Ji et al., 2017 (link)), for at least 5 min prior to determining the intracellular pH (pH_i). pH_i was determined by measuring BCECF fluorescence using dual excitation wavelengths (495 and 440 nm) and an emission wavelength (530 nm). NBC activity was measured by incubating the cells with a CO₂-saturated bicarbonate-buffered as described previously (Ji et al., 2017 (link)) containing sodium hydrogen exchanger inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 1154-25-2, Sigma Aldrich), followed by acidification with Na⁺-free bicarbonate-buffered medium. Images were obtained with a CCD camera (Q-Imaging) and analyzed with a Meta Fluor system (Molecular Devices). The images were individually normalized by subtracting the background fluorescence signal from the raw background signals.

Cells were attached onto coverslips one day before and loaded in the chamber with 4 μM BCECF-AM (2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, 0061, TEFlabs Inc, Austin, TX, USA) in the presence of same volume of 0.05% pluronic acid (P-3000MP, Invitrogen, Carlsbad, CA, USA) for 15 min at RT. After incubation of the fluorescence dye, the cells were perfused with regular solution, as previously described [24 (link)], for at least 5 min prior to intracellular pH (pH_i) measurements. The pH_i was measured by BCECF fluorescence using dual excitation wavelengths of 495 and 440 nm and emission wavelength of 530 nm. The cells were incubated with CO₂-saturated HCO₃⁻ solution for the acidification of the cytosol, and then, perfused with Cl⁻-free HCO₃⁻ solution. All incubated solutions were maintained at 37°C. Cl⁻-HCO₃⁻ exchanger (CBE) activity of AE2 was calculated from the slope of increase in pH_i during the first 30-45 sec in Cl⁻-free HCO₃⁻ solution and expressed as the percent fold change relative to that of CBE activity of control. Fluorescence images were obtained with Retiga 6000 CCD camera (Q-Imaging) recruited to an inverted microscope (Olympus, Tokyo, Japan) and analyzed with a Meta Fluor system (Molecular Devices, San Jose, CA, USA). Each image was analyzed after the removal of the background fluorescence.

Cells were incubated in 2 mM Ca²⁺ Ringer’s solution containing 5 μM Fluo-4 AM (F14201; Invitrogen) and 0.1% pluronic acid (P3000MP; Invitrogen) to permeabilize cells at 21°C for 30 min, followed by deesterification in Fluo-4-free Ringer’s solution for 30 min. Cells were bathed in 2 mM Ca²⁺ Ringer’s solution and excited by a 488 nm laser and the resulting fluorescence was monitored using an inverted microscope with a Plan-Apochromat 40×/1.40 oil objective, connected to an Andor W1 spinning-disk confocal with a Photometrics Prime 95B camera. Image acquisition occurred at 21°C every 5 s using Micromanager software. At 100 s, cells were perfused with 100 μM UTP or bradykinin in 2 mM Ca²⁺ Ringer’s solution for 100 s. At 200 s, cells were perfused with 2 mM Ca²⁺ Ringer’s solution without G_q agonist and imaged until 400 s. Images stacks were analyzed in ImageJ. An ROI was drawn in the cytosol of Fluo-4-loaded cells and measured for fluorescence. Intensity was normalized to minimum intensity value before G_q agonist application. Amplitude and area under the curve measurements were made by GraphPad Prism.