| Assay Method Information | |
| | NK-1, NK-2 and NK-3 Calcium Flux FLIPR assays |
| Description: | Experimental procedure for NK-1, NK-2 and NK-3 calcium flux FLIPR assay: Perform the assay in the following steps: 1) Cell preparation: thaw each cell (NK-1, NK-2 and NK-3) in 37° C. water bath with gentle shaking, transfer cell suspensions to 50 mL conical tubes and add plating media to 45 mL mark. Count cells using a ViCell for concentration. Re-suspend the cells in the growth media to a concentration of 10×105 per mL. Add 20 μL per well of the cell suspension to the 384-well plates (20,000 cells/well). Place the cells at 37° C. 5% CO2 incubator overnight. 2) Calcium Flux FLIPR assay: a) Prepare Probenecid reagent by adding 1 mL FLIPR Assay Buffer to 77 mg probenecid to make 250 mM solution. b) Prepare assay reagent (2×8 μM Fluo-4 Direct Loading Buffer): Thaw one vial of Fluo-4 Direct crystals, add 10 mL of FLIPR Assay Buffer to the Vial; add 0.2 mL of Probenecid to each 10 mL vial of Fluo-Direct. c) Compounds preparation: The compounds are serially diluted in 100% DMSO 1:3 for 10 pts by Echo. Then dispense 900 nL of compounds to the 384-compound plate. Remove cell plate from incubator and gently dispense 20 μL of 2×Fluo-4 Direct to 384 well cell culture plate. Incubate for 50 min at 37° C. 5% CO2 and 10 min at room temperature. Remove cell plate from incubator and place it into FLIPR (Molecular Devices). Place compound plate and tip box into FLIPR. For the dose response curve (DRC) plate: a) Run the Protocol on FLIPRTETRA. b) Transfer 10 μL of assay buffer from 384-well plate to the cell plates. c) Read fluorescence signal. d) Transfer 10 μL of the compounds from the DRC plate to the cell plates. e) Read fluorescence signal. f) Calculate the Max-Min starting from Read 90 to Maximum allowed. Calculate the EC80 values for each cell line using FLIPR. g) Prepare 6×EC80 concentrations of agonist reference compounds. For the compound plate in antagonist test: a) Run the Protocol on FLIPRTETRA. b) Transfer 10 μL of references and compounds from the compound plate to the cell plates. c) Read fluorescence signal. d) Transfer 10 μL of 6×EC80 concentrations of agonist reference compounds to the cell plates. e) Read fluorescence signal. f) For antagonist test, calculate the Max-Min starting from Read 90 to Maximum allowed. h) Analyze the data using GraphPad Prism 5.0. |
| Affinity data for this assay | |
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