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28 protocols using Fluo-8

Calcium flux was measured using a Fluo-8 assay (AAT Bioquest, Sunnyvale, CA, USA) to assess the effect of bac I and II, either alone or in combination, on cytosolic Ca2+ in HT-29, 2H-11, and HUVEC, as per the manufacturer’s instruction. Briefly, cells were seeded in the respective complete medium at 2 × 104 cells per well on 96-well flat-bottomed plates and incubated overnight. Cells were labelled with a dye working solution consisting of Hank’s and HEPES Buffer (HHBS), supplemented with 0.08 % pluoronic® F-127 (AAT Bioquest), 4 mM probenecid (AAT Bioquest) and 4 µM Fluo-8. Pluoronic® F-127 and probenecid were used to improve aqueous solubility of AM esters attached to Fluo-8 and to reduce the leakage of the de-esterified indicators, respectively. The plates were incubated for 30 min at 37 °C, followed by a 30-min incubation at room temperature. Media was replaced with HHBS containing 1 mM probenecid and vehicle or bac I and/or II, and fluorescence was measured at time 0, 10 and 30 min, 1, 1.5, 2, 3 and 6 h on a FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany).
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We used acetoxymethyl (AM) ester forms of the following organic Ca2+- dyes (suppliers and catalog numbers listed in parentheses): Cal-520 (AAT Bioquest #21130), Fluo-8 (AAT Bioquest #21083), Fluo-8 high affinity (AAT Bioquest #21091), Fluo-8 low affinity (AAT Bioquest #21097), Rhod-4 (AAT Bioquest #21122) ; Fluo-4 (Invitrogen #F-14201), Oregon Green BAPTA-1 (Invitrogen #O-6807), X-Rhod-1 (Invitrogen #X-14210); and Asante Calcium Red (TefLabs #3010). All dyes were reconstituted with dimethyl sulfoxide (DMSO) containing 20% pluronic F-127 (DMSO/F-127; Invitrogen; #P-3000MP) to a final concentration of 1 mM and were stored, shielded from light, at −20°C. The membrane-permeant caged IP3 analogue ci-IP3/PM [D-2,3-O-Isopropylidene-6-O-(2-nitro-4,5 dimethoxy) benzyl-myo-Inositol 1,4,5-trisphosphate Hexakis (propionoxymethyl) ester] was purchased from siChem (#cag-iso-2-145-10), solubilized with DMSO/F-127 to a final concentration of 200 μM and stored at −20°C. The GCaMP6 -slow, -medium, and -fast variants (plasmids #40753, #40754, and #40755, respectively) were obtained from Addgene. Lipofectamine 2000, for GCaMP6 induction, was purchased from Invitrogen (#11668030). All other reagents were purchased from Sigma-Aldrich.
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MDCK II cells were seeded in 96-well plates at a density of 3.4 × 104 cells per well. The cells were cultured for 3 days, and the medium was changed every day to establish monolayer integrity. After specific treatments, monolayers were washed with Hank’s balanced salt solution (HBSS) and treated with Fluo-8 loading buffer (5 μM Fluo-8 [#21080, AAT Bioquest, Sunnyvale, CA], 2.5 mM probenecid [#ALX-430-113-G005, Cosmo Bio, Tokyo], 1% F-127 [#59004, Biotium, Hayward, CA] in HBSS) at 37 °C for 1 h. Cells were washed with HBSS twice, and recording buffer (2.5 mM probenecid in HBSS) and various compounds were added. The Fluo-8 fluorescence was measured every 3 min for 60 min with a PowerscanHT fluorescence microplate reader (Dainippon Sumitomo Pharma, Osaka, Japan) at the excitation and emission wavelengths of 485 and 528 nm, respectively.
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At first, rat basophil leukemia cells were suspended in HBSS buffer (Invitrogen) complemented with 20 mM Hepes, and distributed in microplates at a density of 2.768 × 104 cells/well. Thereafter, a mixture of fluorescent probe (Fluo8, AAT Bioquest) and probenecid in HBSS buffer (Invitrogen) complemented with 20 mM Hepes (Millipore) (pH 7.4) was added and plates were incubated for 60 min at 30 °C. Then, the assay plates were positioned in a FlipR Tetra microplate reader (Molecular Device, San Jose, CA, USA) for the addition of the test compound, reference agonist/antagonist, or HBSS buffer (basal control). Change in fluorescence intensity that varies proportionally to the free cytosolic Ca2+ ion concentration was measured.
For stimulated control measurements, 1 µM DPDPE was added in separate assay wells. The results are expressed as a percent of the control response to DPDPE at 1 µM or a percent inhibition of the control response to DPDPE at 25 nM. The standard reference agonist and antagonist were DPDPE and naltriben mesylate, respectively.
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Kinetic measurements of transient intracellular calcium mobilization in NPCs were performed in poly-L-ornithine/laminin (Sigma–Aldrich) coated black 96-well clear-bottom plates (Greiner Bio-One). Cells were seeded at a density of 3.0 × 105 cells per ml and incubated for 24 h. The following day, the medium was removed and cells were loaded with 4 µM Fluo-8 (AAT Bioquest) as the indicator dye, reconstituted in a modified Tyrode’s assay buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 135 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 10 mM glucose adjusted to pH = 7.4.) and incubated for 30 min at 37 °C and 5% CO2, and another 30 min at room temperature in the dark [27 (link),28 (link)]. Then, the Fluo-8 loading buffer was removed and replaced with fresh Tyrode’s assay buffer. The agonists, diluted in Tyrode’s assay buffer, were added automatically using the injector unit from Tecan Infinite M1000 Pro microplate reader Tecan (Maennedorf, Switzerland). After 16 s of baseline measurements, the compound was added, and fluorescence was measured for 80 s using excitation at λ = 490 nm and emission at λ = 525 nm. Responses were measured as the maximal peak height in relative fluorescent units (RFUs), and the maximum fluorescence signal was generated with the calcium ionophore A23187 (Sigma–Aldrich).
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HL-1 cells were passaged and seeded onto an imaging dish with a polymer coverslip bottom (Ibidi) and allowed to adhere. Fluo-8 (490/525; AAT Bioquest) was added into the medium (final concentration 2 µM; stock solution in DMSO 2 mM), and the cells were incubated for 20 min at 37°C. After the incubation, the solution was replaced with fresh medium, and the cells were placed on the heated stage (37°C) of an inverted microscope. Ca2+ images in the line-scan (line size 3.37 µm, 100 Hz) mode were recorded using a laser scanning confocal microscope Olympus FL1200 (Olympus) with a ×40 water immersion objective, in the x–y mode. Kinetic properties of intracellular Ca2+ were analyzed via the in-house Python-based script (Kabanov, 2021 ).
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Islets were loaded with Fluo 8 (AAT Bioquest, cat. no. 20494) in HEPES‐bicarbonate buffer containing (in mmol/L) 120 NaCl, 4.8 KCl, 24 NaHCO3, 0.5 Na2HPO4, 5 HEPES, 2.5 CaCl2, 1.2 MgCl2 and supplemented with 8 mM D‐glucose. Ca2+ fluxes were measured using a spinning disk microscope comprised of a Nikon Ti‐E frame, 10×/0.3 NA air objective, North 89 LDI laser bank and CrestOptics V2 X‐light spinning disk unit. Excitation was delivered at λ = 470 nm, with emission collected at λ = 500–550 nm. Intracellular Ca2+ traces were normalized as F/Fmin, where F is fluorescence at any given time point, and Fmin is mean minimum fluorescence. To calculate photoswitching efficiency for each compound, AUC was calculated at each timepoint and then normalized to values before illumination. JB253, JB1793, JB1794, JB1795 and glimepiride were applied to islets at 50 μM.
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Measurement of [Ca2+]c was performed by using a fluorescent Ca2+ indicator Fluo-8 (AAT Bioquest, Sunnyvale, CA). MIN6 cells and isolated β-cells were loaded with 2 μM Fluo-8/ AM dissolved in HBSS for 20 min at room temperature. To monitor [Ca2+]c using Cameleon-nano15, MIN6 cells were transfected with PM-Cameleon-nano15 [32 (link)] by electroporation as described previously [27 (link)]. The cells were placed on a 35 mm glass bottom culture dish. The cells were visualized with an Olympus UPlanAPO 10x Water Objective lens (Olympus, Tokyo, Japan). To detect fluorescence images, we used AQUACOSMOS/ASHURA, 3CCD based fluorescence energy transfer imaging system (Hamamatsu Photonics, Hamamatsu, Japan). Fluo-8 fluorescence was obtained by a U-MGFPHQ cube (Olympus), and expressed as the ratio of cytosolic fluorescence and initial intensity (F/F0). PM-Cameleon-nano15 fluorescence was obtained by a 440AF21 excitation filter (Omega Optical, Brattleboro, VT) and DM455DRLP dichroic mirror (Omega optical) and expressed as the ratio of CFP/YFP. These Images were captured with at a C7780-22 ORCA3CCD camera (Hamamatsu Photonics) at 10-second intervals.
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Cells were differentiated on Matrigel-coated 96-well plates (Greiner). At day 9 of induction, cultured cells were loaded with Fluo-8, a fluorescent Ca2+ indicator (AAT Bioquest) and incubated at 37 °C for 30 min. The detection of Ca2+ through electric stimulation and the analyses were performed with an imaging plate reader FDSS/μCELL system (Hamamatsu photonics). Electric stimulation was applied with electrical field stimulation (EFS) of 96 multi-electrodes at 12 V with a 50 ms interval and a 0.2 Hz mono phase for 1 min after 5 s of resting phase. The sampling rate was set to 30 ms and fluorescence excitation was set at 480 nm with an LED excitation light source. Emission was set at 540 nm using an emission filter and detected with an electron multiplying CCD (EMCCD) camera. Measurements for 96 wells were performed under uniform conditions at 37 °C with simultaneous stimulation and detection.
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hiPSC-CMs at day 23 were seeded onto EZ Sphere (AGC Techno Glass, Shizuoka, Japan) at 5 × 104 cells/cm2 to form a spheroid. The following day (day 24), spontaneous beating was confirmed, and the spheroids were moved to a normoxic (20% O2) or hypoxic (1% O2) incubator for 4 or 12 days to be cultured in DMEM supplemented with 10% FBS and penicillin–streptomycin (Sigma–Aldrich). The culture medium was changed every 4 days. To investigate intracellular Ca2+ transients, spheroids were transferred to 6-well plates and loaded with 5 μM Fluo-8 (AAT Bioquest, Sunnyvale, CA, USA) in DMEM supplemented with 10% FBS for 1 h in a humidified incubator with 5% CO2 at 37 °C. Ten minutes before observation, each well was immersed in phenol red-free DMEM (Nacalai Tesque, Kyoto, Japan) containing 10% FBS and penicillin–streptomycin. Changes in fluorescence levels were monitored at a rate of 100 ms with an ORCA-R2 CCD camera (Hamamatsu Photonics K.K., Shizuoka, Japan) and processed with Aquacosmos image processing software (Hamamatsu Photonics K.K.) for baseline correction and colored visualization. The Ca2+ transient was recorded during electrical stimulation at 1 Hz (C-Pace EP, IonOptix, Westwood, MA, USA).
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