Fura 2 am

The intracellular Ca²⁺ concentration, [Ca²⁺]_I, in drug-treated OUMS-27 cells, was measured by Fura-2 AM, a calcium-sensitive dye (Dojindo, Kumamoto, Japan). Briefly, OUMS-27 cells were loaded with 5 μM Fura-2 AM in loading buffer containing 0.01% pluronic F-127 (Dojindo) for 30 min at 37 °C. After washing Fura-2 AM out of the loading buffer, the relative transient calcium concentration (OD_340nm/OD_380nm excitation ratio) was recorded before and after the addition of 10 mM GABA (Sigma-Aldrich) in a perfusion chamber using the AQUACOSMOS/RATIO, C7773 (Hamamatsu photonics, Hamamatsu, Japan). Recording was continued after washing the 10 mM GABA out of the buffer. [Ca²⁺]_I after pretreatment with 1 μM CGP and application of 10 mM GABA was recorded using a similar method.

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured by ratiometric fura-2 microfluorometry in combination with digital imaging, as previously reported (Kohno et al., 2003 (link), 2007 (link)). Briefly, after incubation with 2 μM fura-2/AM (Dojindo, Kumamoto, Japan, F016) for 45 min, the cells were mounted in a chamber and superfused with HKRB at 1 ml/min at 33°C. Fluorescence images due to excitation at 340 and 380 nm were detected every 10 s with a cooled charge-coupled device camera (ORCA-R2 C10600, Hamamatsu Photonics, Hamamatsu, Japan), and the ratio image was produced by an Aquacosmos (Hamamatsu Photonics). The data were obtained from single cells identified as neurons by previously reported procedures (Kohno et al., 2003 (link), 2007 (link)); briefly, they have a relatively large diameter (≥10 μm), and their cell bodies are clear and round on phase-contrast microscopy. Cells with astrocyte-like flat morphology were excluded. The data were obtained from cells that met these criteria for neurons.

The transfected HEK293 cells were seeded on glass coverslips (Matsunami) coated with poly-L-lysine solution (Sigma-Aldrich) then incubated for 4–10 h. HEK293 cells or cultured neurons were loaded with 5 µM Fura-2 AM (Dojindo) for 15–20 min in the growth medium. Fura-2 fluorescence was measured in HBS (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 2 mM CaCl₂, 11.5 mM glucose, and 20 mM HEPES, pH 7.4). Fluorescence images were obtained using a fluorescence microscope (IX71, Olympus) equipped with a complementary metal-oxide semiconductor (CMOS) camera (ORCA-flash 4.0, Hamamatsu Photonics) under xenon-lamp illumination, and analyzed with a video imaging system (AQUACOSMOS, Hamamatsu Photonics) according to the manufacturer’s protocol. The ratio of 340:380 nm fluorescence was determined from the images, on a pixel-by-pixel basis. To facilitate the screening assay in HEK293 cells, three different cell lines that expressed one of the constructs were co-cultured on a glass coverslip. Each mutant can be distinguished by co-transfected fluorescent proteins that have distinct colors as a marker, and the glutamate responses of three different mutants were assayed simultaneously. The Δratio was defined as the difference between the maximum and the initial ratio values. The Δratio was fitted with KaleidaGraph using following equation (1): a + (b-a)/(1 + (x/c)^d), and the EC₅₀ value was calculated.

The transfected HEK293 cells were seeded on glass coverslips (Matsunami) coated with poly-L-lysine solution (Sigma) and incubated for 4 h at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The calcium indicator Fura-2 AM (Dojindo) was loaded to the cells at 5 μM for 20–30 min. The imaging experiment was carried out in HBS buffer (20 mM HEPES pH 7.4, 107 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 2 mM CaCl₂, 11.5 mM glucose). The fluorescence images and the Fura-2 ratio were measured using a fluorescence microscope (IX71, Olympus) equipped with a complementary metal-oxide semiconductor (CMOS) camera (ORCA-flash 4.0, Hamamatsu Photonics) under xenon-lamp illumination, and analyzed with a video imaging system (AQUACOSMOS, Hamamatsu Photonics) following the manufacture’s instruction. In imaging experiments, three different HEK293 cells transfected with one of the mGlu1 mutants were co-cultured on a glass coverslip, and each mutant was visually distinguished by the transfection markers. These three different cells were assayed simultaneously. The Δratio value was defined as the difference between the maximum ratio value after adding the reagent (metal ion or complex, or glutamate) and the average ratio before adding the reagent. The Δratio was fitted with KaleidaGraph to calculate the EC₅₀ value using the equation: a + (b-a)/(1+(x/c)^d).

Naive CD4⁺ T cells isolated from PB of healthy donors were suspended at 3 × 10⁶ cells/ml in Hank's balanced salt solution (HBSS) containing 1 mg/ml of bovine serum albumin (BSA) and 10 mM HEPES, pH 7.4 (HBSS-BSA) and incubated with 1 mM Fura-2-AM (Dojindo, Kumamoto, Japan) at RT for 30 min in the dark. After washing twice with HBSS-BSA, cells were suspended in HBSS-BSA at 2.5 × 10⁶ cells/ml. 2 ml of the cell suspension in a quartz cuvette was placed in a luminescence spectrometer (RF-5000, Shimadzu, Kyoto, Japan) and fluorescence was monitored at an emission wavelength of 510 nm, and excitation wavelengths of 340 and 380 nm every 20 ms. Calibration of fluorescence in terms of [Ca²⁺]i was calculated from the ratio 340/380 excitation fluorescent values.

Changes in intracellular calcium were measured with the fluorescent calcium indicator, Fura-2, as previously described (Masuoka et al., 2015 (link), 2017 (link)). For the microscopic fluorometric measurement, cultured trigeminal ganglion neurons were washed twice with Krebs–Henseleit solution and incubated for 1 h in a solution containing 3 μM of Fura-2-acetoxymethyl ester (Fura-2 AM; Dojindo Laboratories, Kumamoto, Japan) and 0.005% Cremophor EL (Sigma–Aldrich). After incubation, the cells were washed in Krebs–Henseleit solution for 30 min, and culture dishes were placed on the stage of an inverted microscope (ECLIPSE TE 300, Nikon, Tokyo, Japan) equipped with a 20× S-fluor objective. Fluorescence images were recorded and analyzed using a video image analysis system (HCimage, Hamamatsu Photonics, Hamamatsu, Japan). The experimental agents were dissolved in Krebs–Henseleit solution and delivered by continuous perfusion in the recording chamber with a peristaltic pump (2 ml/min). The perfused solutions were maintained at 34°C with a temperature controller (TC-344C and SH-27B, Warner Instruments). Image pairs of Fura-2 fluorescence were captured every 10 s at an emission wavelength of 510 nm by exciting Fura-2 at 340 and 380 nm. The 340–380 nm fluorescence ratio (F340/F380) was used as a parameter of intracellular calcium concentration.