( br Step 5. ...)High-content screening of GPCR activation with MetaXpress, AcuityXpress and the Transfluor assay system

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Step 5. Incubate at 37C, 5% CO₂ for 30 minutes.

Step 6. Add 10 L of the 175 nM isoproterenol stimulation solution or ascorbic acid media (unstimulated control) to each well.

Step 7. Incubate at 37C, 5% CO₂ for 4045 minutes (vesicle formers) or 30 minutes (pit formers).


Sample fixing and staining
Step 1. Stop assay by adding 35 L 4% methanol-free formaldehyde to each well. Cover and incubate for 45 minutes at room temperature.

Step 2. Wash each well with 50 L DPBS.

Step 3. Add 8 L of 0.05 mg/mL DAPI to each well. Incubate at room temperature for 15 minutes.

Step 4. Rinse wells twice with 75 L DPBS, leaving in 75 L DPBS.

Step 5. Cover plate with a plate seal and store at 4C, protected from light, until image acquisition.


Image acquisition
Step 1. Using MetaXpress, acquire unbinned images on an ImageXpress 5000A or Discovery-1 imaging system using DAPI and FITC filter sets and a 20x Plan Fluor or Plan Fluor ELWD objective. The results described here were obtained from imaging two sites per well.


Image analysis
Step 1. In MetaXpress, retrieve your images of interest from the database and analyze them with the Transfluor assay module. Select the appropriate source images, minimum and maximum widths, and intensity above local background for both the pit/
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