| Assay Method Information | |
| | BLT-1 cAMP Assay |
| Description: | The ability of compounds to antagonize the human BLT1 receptor was determined using a kit to measure changes in intracellular cyclic AMP levels (cAMP dynamic assay kit, Cisbio Cat. No. 62AM4PEC). HEK293 cells recombinantly expressing human BLT1 receptor, previously frozen in Recovery Medium (Life Technologies, Cat. No. 12648-010) were thawed and diluted into assay medium (HBSS (Hyclone SH 30268.01), 20 mM HEPES (Gibco 15630-106), 800 μM IBMX (Sigma I5879), 0.1% DTPA BSA (Perkin Elmer CR84-100)). The cell suspension was centrifuged at 200×g for 10 min and then resuspended in fresh assay medium to a density of 2.5×105 cells/mL. A Labcyte Echo 550 acoustic dispenser was used to transfer 25 nL of test compound dissolved in DMSO into the wells of a dry 384-well plate (Greiner 784075). All subsequent liquid additions were performed using a BIORAPTR (FRD; Beckman Coulter). Next, 5 μL of cell suspension was added and incubated for 20 min. at 37° C. and 5% CO2 in a humidified plastic tray. To test for agonist activity 51 μL of assay buffer containing forskolin (Sigma F-6886. 4 μM for BLT1) was added and incubated for 30 minutes at 37° C. and 5% CO2 in a humidified plastic tray. To test for antagonist activity 5 μL of assay buffer containing forskolin (Sigma F-6886. 4 μM for BLT1) and either LTB4 (Sigma Aldrich L0517; 2 nM BLT1) was added and incubated for 30 minutes at 37° C. and 5% CO2 in a humidified plastic tray.The levels of cAMP were detected using the CisBio kit following the manufacturer's instructions: cAMP-d2 vial was reconstituted with distilled water and then diluted accordingly with conjugate & lysis buffer. 5 μL of cAMP-d2 working solution was added to all of the wells of the assay plate. cAMP-Cryptate vial was reconstituted with distilled water and then diluted accordingly with conjugate & lysis buffer. 5 μL of cAMP-Cryptate working solution were added to the assay plate. The assay plate was shaken for 3 minutes and incubated at room temperature for 45 minutes, then read on Perkin Elmer Envision. cAMP standard curve and fit data were plotted using a 4 parameter dose response curve fitting algorithm to fit curve. A 4-parameter curve fit (Max, min, log EC50 and slope) was used to transform fluorescent 665 nm/615 nm ratio signal to cAMP concentration. After normalization to treated and untreated controls, the percent effect of signal at each compound concentration was calculated. The plot of percent effect versus the log of compound concentration was fit with a 4-parameter concentration response equation to calculate EC50 values. Compound concentrations tested were 10 000, 3 333, 1 111, 370.4, 123.4, 41.2, 13.7, 4.6, 1.5 and 0.5 nM with 0.25% residual DMSO. |
| Affinity data for this assay | |
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