ANO1/TMEM16A and YFP expressing FRT cells were plated in 96-well black-walled microplates (Corning Inc., Corning, NY) at a density of 20,000 cells per well in F12 medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin. Assays were done using FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) and MARS Data Analysis Software (BMG Labtech). Each well of 96-well plate was washed 3 times in PBS (200 μL/wash), leaving 100 μL PBS. Test compounds (1 μL) were added to each well at 25 μM final concentration. After 10 min, 96-well plates were transferred to a plate reader for fluorescence assay. Each well was assayed individually for TMEM16A-mediated I- influx by recording fluorescence continuously (400 ms per point) for 2 s (baseline), then 100 μL of 140 mM I- solution containing 200 μM ATP was added at 2 s and then YFP fluorescence was recorded for 6 s. Initial iodide influx rate was determined from the initial slope of fluorescence decrease, by nonlinear regression, following infusion of iodide with ATP.
Black walled microplates
Manufactured by Corning
Black-walled microplates are a type of lab equipment used for various applications. They feature a black-colored wall structure that helps to minimize well-to-well crosstalk and background fluorescence, improving the accuracy of optical measurements. These microplates are designed to accommodate a wide range of sample volumes and are compatible with standard laboratory equipment and instrumentation.
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2 protocols using black walled microplates
1
TMEM16A-mediated Iodide Influx Assay
2
Cell Growth Inhibition Assay
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Cells were plated in black-walled microplates (Corning). At 16 to 24 h after plating, the medium was replaced with drug-containing medium. A 10 μΜ starting concentration was used for all drugs except trametinib (1 μΜ), with 9 serial dilutions of each drug prepared at 1-in-3 ratios. Each dilution was used in four replicates. All of the plates contained media-only and solvent-only controls.
Nuclei were counted 6 d after treatment as a measure of the drug effect on cell growth. The cells were fixed using 4% paraformaldehyde (ProSciTech) in phosphate-buffered saline (PBS) for 10 min and then stained with DAPI staining solution (1 μg/mL DAPI, 50 mM Tris, pH 7.5, 0.2% Triton X-100) for 20 min. Nuclei were imaged using a Cellomics ArrayScan automated microscope (Cellomics, Life Technologies).
Nuclei were counted 6 d after treatment as a measure of the drug effect on cell growth. The cells were fixed using 4% paraformaldehyde (ProSciTech) in phosphate-buffered saline (PBS) for 10 min and then stained with DAPI staining solution (1 μg/mL DAPI, 50 mM Tris, pH 7.5, 0.2% Triton X-100) for 20 min. Nuclei were imaged using a Cellomics ArrayScan automated microscope (Cellomics, Life Technologies).
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