A membrane potential FLIPR assay was developed and validated using a Molecular Devices FLIPR TETRA in 384‐well plate format. Single‐point screening (10 μmol·L–1) of a repurposing library of 1320 compounds identified activators and potentiators of the WT GABRA3/GABRB3/GABRG2 GABAA receptor using the clonal HEK293 cell line expressing this receptor. A voltage‐sensitive fluorescent dye (R8042, Molecular Devices) was loaded into cultured cells. Cells were washed and then incubated in test compound for 4 minutes followed by addition of GABA to a concentration of 15 nmol·L–1 (near the EC20 for GABA stimulation for this assay). Percentage potentiation was measured and compared to the maximum response (15 nmol·L–1 GABA and 1 μmol·L–1 diazepam). Potency determination with eight‐point CRCs in half‐log dilution steps starting at 30 μmol·L–1 was performed for selected compounds. The EC50 of reference compounds diazepam and phenobarbital in this assay was 28 nmol·L–1 and 90 μmol·L–1, respectively.
R 8042
Manufactured by Molecular Devices
The R-8042 is a laboratory equipment product designed for general use in research and testing environments. It serves as a core functional component without any specific intended application. A detailed description cannot be provided while maintaining an unbiased and purely factual approach.
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3 protocols using r 8042
1
GABAA Receptor Potentiation Screening
2
Fluorescent Membrane Potential Imaging
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Cells were seeded onto Matrigel-fibronectin coated 8-well chambered cover glasses (Lab-Tek, Naperville, IL) and incubated overnight in starvation medium, followed by incubation in 400 μL of buffer P (135 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl2, 0.5 mM MgSO4, 0.5 mM Na2HPO4, 10 mM HEPES, 5 mM NaHCO3, pH 7.4) containing 2.8 mM glucose for 2 hours. A vial from a FLIPR membrane potential assay kit, explorer format component A, containing a proprietary plasma membrane potential (Δψp) indicator (“PMPI”) (R-8042; Molecular Devices, Sunnydale, CA) was reconstituted in 10 mL water, and 4 μL added to the incubation immediately prior to imaging as described previously [26 (link),27 (link)]. Excitation was performed at 514 nm and emission recorded with a 530 nm long-pass filter [26 (link)] on a Zeiss LSM510 inverted confocal fluorescence microscope.
3
Membrane Potential Monitoring in INS-1 Cells
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The membrane potential was monitored in INS-1 832/13 cells with the indicator PMPI (R-8042; Molecular Devices, Sunnydale, CA), it is loaded into the cells for 30 min as recommended by the manufacturer. After loading with the dye, the cells were superfused at 1 ml/min (32 °C) with Krebs buffer containing PMPI supplemented as indicated. The fluorescence from PMPI was monitored at excitation wavelength 514 nm and the emitted light signals were read at 530 nm long-pass filter on confocal microscopy (LSM 510, Carl Zeiss, Germany).
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