| Assay Method Information | |
| | FLIPR-3 Calcium Assay |
| Description: | The assay is a Ca2+ mobilization assay that measures changes in intracellular Ca2+ with a FlexStation II scanning fluorometer using a FLIPR-3 Calcium Assay Kit (Molecular Devices, Sunnyvale, Calif.). The procedure is as follows: 1. Human neutrophils were suspended in HBSS− (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1×107 cells in total volume 1.7 mL). 2. Cells were aliquoted (200 μL of the cell suspension per tube, 8 tubes total) and 2 μL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 μL of DMSO (1% final concentration) were added to other 2 tubes. 3. Cells were incubated for 30 min at 37° C. 4. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and cell pellet was re-suspended in 200 μL of HBSS+ (with Ca+ and Mg2+) containing 10 mM HEPES. 5. The compound or DMSO (control) was added again at the same concentrations that were used during cell loading. 6. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 μL (105 cells/well). The Compound Plate contained physiologic agonist (GROα in HBSS−) or HBSS− (control). 7. After 15 sec of reading the basal level of fluorescence using the FlexStation II instrument, 10 μL of GROα or HBSS− were automatically transferred from the Compound Plate into the Reading Plate (final concentration of GROα was 25 nM). 8. Changes in fluorescence were monitored (λex=485 nm, λem=525 nm) every 5 s for 240 to 500 s at room temperature. 9. The maximum change in fluorescence, expressed in arbitrary units over baseline (Max-Min) was used to determine the GROα response. The effect of each compound on the GROα response was normalized and expressed as a percent of the DMSO control, which was designated as 100% response. Curve fitting and calculation of the compound inhibitory concentration that reduces the level of the GROα response by 50% (IC50), or the compound agonist concentration that increases the level of the calcium release by 50% of its maximum induced change (EC50) in the absence of GROα were determined by nonlinear regression analysis of the dose-response curves generated using Prism 4 (GraphPad Software, Inc., San Diego, Calif.). |
| Affinity data for this assay | |
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