Intracellular calcium mobilization was measured by using FLIPR Tetra System (Molecular Devices, Sunnyvale, CA, USA) in accordance with manufacturer’s instruction. Briefly, HEK-hKOR and HEK-hMOR (provided by Professor Ki Duk Park, Korea Institute of Science and Technology) were seeded in 96-well clear-bottom black plate in 100 μL culture medium at a density of 30,000 cells/well and then incubated in a 5% CO2, 37 °C incubator overnight. After incubation, cells were added with 100 μL of the calcium dye dissolved in loading buffer (1 × HBSS plus 20 mM HEPES buffer) for 2 h at 37 °C. Subsequently, the plate was transferred to the FLIPR Tetra System for treating 50 μL of the 5× compounds diluted in loading buffer and reading intracellular calcium mobilization. The final concentration of DMSO was 0.1%. Fluorescence was measured at 1 s intervals for 60 s by using a cooled CCD camera with 470–495 nm excitation LED module and 515–575 nm emission filter and fluorescence signal of calcium mobilization was used as the maximum value minus minimum. A dose response curve was generated with the changes in Relative Fluorescence Units (∆RFU) versus Log10 dose.
Flipr tetra system
Manufactured by Molecular Devices
Sourced in United States
The FLIPR Tetra system is a high-throughput cellular screening platform designed for drug discovery and cell-based assays. The system utilizes fluorescence-based detection to measure cellular responses, such as changes in intracellular calcium levels, in a multiwell plate format. The FLIPR Tetra system provides researchers with a powerful tool for analyzing cellular function and signal transduction pathways.
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Lab products found in correlation
Spectramax i3, by Molecular Devices (2 mentions)
Vx 809, by Selleck Chemicals (2 mentions)
R and s vx445, by MedChemExpress (2 mentions)
Ac1 x281602, by Abbvie (2 mentions)
Ac2 1 x281632, by Abbvie (2 mentions)
Ac2 2 x300549, by Abbvie (2 mentions)
Ap2 x300529, by Abbvie (2 mentions)
Vx 661, by Selleck Chemicals (2 mentions)
Vx 770, by Selleck Chemicals (2 mentions)
Calcium 6 dye, by Molecular Devices (2 mentions)
Flipr calcium 5 assay kit, by Molecular Devices (2 mentions)
Fugene hd, by Promega (1 mentions)
Probenecid, by Merck Group (1 mentions)
Somatostatin 14, by Bachem (1 mentions)
Fluo 4 am, by Abcam (1 mentions)
Earlytox cardiotoxicity kit, by Molecular Devices (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
Prism 7, by GraphPad (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
17 protocols using flipr tetra system
1
Intracellular Calcium Mobilization Assay
2
Calcium Imaging of Glutamate Responses
3
FLIPR Calcium Flux Assay Protocol
4
Measuring CFTR-mediated Chloride Conductance
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Apical CFTR-mediated chloride conductance was measured as previously described18 (link). Briefly, hPSC-derived cholangiocytes were grown in 96-well plates and treated for 24 h with either or combination of CFTR modulators, 3 μM VX809, 3 μM VX661 (Selleck), 3 μM R and S-VX445 (MedChemExpress), 0.5 μM AC1 (X281602), 3 μM AC2-1 (X281632), 3 μM AC2-2 (X300549) (Abbvie), or DMSO. Cells were labeled using blue membrane potential sensitive FLIPR dye dissolved in sodium and chloride-free buffer (150 mM NMDG, 150 mM gluconolactone, 10 mM HEPES, pH 7.4, 300 mOsm) at a concentration of 0.5 mg/ml. Cells were incubated for 30 min at 37 °C. The plate was read in a fluorescence plate reader (FLIPR® Tetra System or SpectraMax i3; Molecular Devices) at 37 °C. After reading baseline fluorescence, CFTR was stimulated with a combination of the cAMP agonist Forskolin (10 μM) and potentiators, 1 μM VX770 (Selleck) or 1.5 μM AP2 (X300529) (Abbvie). CFTR-mediated depolarization was detected as an increase in fluorescence, and repolarization was detected as a decrease with addition of 10 μM CFTR-specific inhibitor CFTRInh-172 to all wells.
5
Calcium Oscillation Assay for Neural Functionality
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Calcium oscillation assays were performed as described in our previous publication [9 (link)]. Prior to compound addition, cells were loaded with Calcium 6 dye (Molecular Devices, San Jose, CA, USA) according to the manufacturer’s specifications. To assess the impact on neural functionality, compounds were added and incubated for 30 min before the assay. All recordings (2D and 3D) were done in a FLIPRTETRA system (Molecular Devices, San Jose, CA, USA). For induced activity (2D), cells were challenged with glutamate or kainate, both at 100 μM. For 3D cultures, neural spontaneous activity was recorded for 10 min. Data were analyzed using Peak Pro software (Molecular Devices, San Jose, CA, USA).
6
Gα16gust44 Receptor Activation Assay
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The HEK293T–Gα16gust44 cells were loaded with the fluorescence dye Fluo-4 AM (Abcam, Cat# ab241082), in combination with 2.5 mM probenecid (Sigma Aldrich, Cat# P8761) for 1 h in the dark at culture conditions as reported previously [9 (link), 34 (link)]. Briefly, the cells were washed with C1 buffer and incubated for half an hour in the dark at room temperature. Directly before the measurement, the plate was washed again with C1 buffer. To apply agonists and to detect fluorescence changes a FLIPRTETRA system (Molecular Devices, San Jose, USA) was used. As a cell viability control, somatostatin 14 (Bachem, Cat# 4033009) at a final concentration of 100 nM was employed. For calculations of ΔF/F values, the fluorescence changes observed for receptor transfected cells were mock subtracted using the corresponding identically treated cells transfected with empty vector. The data from three independent experiments, each performed in duplicates were checked for statistical significance using student’s t-test (two-sided; p < 0.05).
7
Calcium Signaling Assay for GPR142
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HEK293-hGPR142 or HEK293-mGpr142 cells were plated in 96-well plates at 50000 cells per well and allowed 24 hours for attachment. After addition of Fluo-5 dye (Calcium-5 Assay Kit, Molecular Probes), cells were incubated for 1 hour at room temperature. Dye was then removed and substituted with assay buffer (HBSS with Ca/Mg, 20mM HEPES, 0.1% BSA). Compounds were added to plates, and signal was quantified by FLIPR tetra system (Molecular Devices).
8
Calcium Transient Measurement Protocol
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Calcium transients were assessed using an EarlyTox Cardiotoxicity Kit (Molecular Devices, San Jose, CA, USA) (28 (link)). Calcium dye loading was performed according to the manufacturer’s instructions. EarlyTox calcium dye was resuspended in the supplied buffer and added to the cells in a 1:1 ratio with the CM maintenance media. The plate was incubated for 2 h (37°C, 5% CO2) before recording calcium transients for 2 min at 37°C on the FLIPR Tetra System (Molecular Devices), using the following parameters: excitation, 470–495 nM; emission, 515–575 nM; exposure time, 50 ms; light-emitting diode intensity, 50%; and interval time, 100 ms. Calcium traces were produced and analyzed using SoftMax Pro Software (Molecular Devices).
9
Measuring FFAR1 receptor activation in CHO cells
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Chinese hamster ovary (CHO) cells stably expressing human FFAR1 with different receptor mRNA expression levels (clones #104 and #2) [32 (link)] were cultured with minimum essential medium-alpha (FUJIFILM Wako) containing 10% dialyzed fetal bovine serum (FBS, GE Healthcare Life Sciences, Buckinghamshire, UK), 10 mM HEPES (Thermo Fisher Scientific, Waltham, MA), 100 IU/mL penicillin, and 100 μg/mL streptomycin in 5% CO2 at 37°C. Cells (1 x 104 cells /well) were seeded in 384 well plates and then incubated overnight. After removing of the medium, cells were incubated in 30 μL of loading buffer (Hanks' Balanced Salt Solution containing 20 mM HEPES, 0.1% fatty acid-free BSA (FUJIFILM Wako), 0.08% Pluronic F127 (#CSK-01F, Dojindo Kumamoto, Japan), 2.5 mmol/L Probenecid (#CSK-03F, Dojindo) and 2.5 μg/mL Fluo4 (#F311, Dojindo)) for 60 min in 5% CO2 at 37°C. Test compounds at various concentrations were added into the cells and increase of the intracellular Ca2+ concentration was monitored by the FLIPR Tetra system (Molecular Devices, Tokyo, Japan) for 180 sec. EC50 was calculated by data analysis using a 4-parameter logistic equation in Graphpad Prism 7 software.
10
Calcium Signaling Assay in CHO-FFA4 Cells
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CHO-FFA4 cells seeded on black-walled 96-well plates (Greiner, USA) at a density of 60 000 cells per well were loaded for 1 h at 37 °C with Calcium-6 dye (Molecular Devices, USA) in Hanks' balanced salt solution containing 20 mM HEPES (pH 7.4). The level of [Ca2+] was then monitored using a FLIPRTetra system (Molecular Devices, USA).
For antagonists to work, AH7614 was first incubated with the cells for 10 min, before the addition of the compounds (i.e. the agonists). Data were expressed as fluorescence (arbitrary units) versus time.
For antagonists to work, AH7614 was first incubated with the cells for 10 min, before the addition of the compounds (i.e. the agonists). Data were expressed as fluorescence (arbitrary units) versus time.
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