EDTA-free protease inhibitor cocktail tablets (Roche 1873580)
Buffer: 25-mM HEPES pH 7.4, 5-mM MgCl2, 1-mM CaCl2, 0.2% (w/v) BSA, protease inhibitors (one tablet per 50-ml buffer).
Note: that BSA and protease inhibitors were added freshly on the day of use.
GraphPad Prism™ software (GraphPad Software)
1. Human recombinant CXCR2 receptor membrane preparation was used in conjunction with [125I]GRO-αligand and WGA PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 125-nM IL-8. The standard assay format was as follows
2. Reagents were added in the following order: Buffer or DMSO solution, unlabeled ligand (NSB wells), labeled ligand, premixed bead, and membrane. Total assay volume was 40 µl
3. Diluted membrane and bead were premixed at room temperature immediately prior to assay addition.
4. Wells contained 10 µl of 1.48-nM [125I]GRO-α(final concentration 0.37 nM) unless otherwise stated. Wells also contained 0.1 mg of beads and 1.2 µg of membrane, which were added together in a 20-µl volume.
5. NSB wells contained 10 µl of 0.5-µM IL-8 (final concentration 0.125 µM) in addition to the above.
6. For DMSO tolerance studies, DMSO was diluted in binding buffer to give final concentrations per well as shown in the results.
7. Plates were sealed and incubated in darkness for 8 h at room temperature (20–25 ºC) unless otherwise stated.
8. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 3 min with quasi-coincident averaging and 3 ×3 binning.