| Assay Method Information | |
| | FLIPR Calcium Assay |
| Description: | Ca2+ influx was measured using a FLIPR calcium assay kit (R8033; Molecular Devices, Sunnyvale, Calif.). The Ca2+ indicator dye was dissolved in Hanks' balanced salt solution supplemented with 20 mM HEPES buffer (HBSS/HEPES) according to the manufacturer instructions. Prior to start of the assay, the medium was removed by aspiration, and cells were loaded with 100 μL Ca2+ dye for 2 to 3 hours at room temperature. AITC (allyl isothiocyanate) was used to activate and open the channels. (4×) solutions of the test compounds were prepared in HBSS/HEPES, and 50 μL were added to the cells at a delivery rate of 10 μL/sec. Changes in fluorescene were measured over time in a fluorometric imaging plate reader (FLIPR), Molecular Devices). Two additions were made over the course of an experimental run. For agonist experiments, assay buffer was added at the 10 s time point, followed by addition of agonist at the 3 minute 10 sec time point. For antagonist experiments, the antagonist was added at the 10 sec time point, followed by addition of the agonist 3 minutes later. Final assay volume for both the agonist and antagonist experiments was 200 μL. Total length of an experimental run was 6.5 minutes. |
| Affinity data for this assay | |
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