| Assay Method Information | |
| | Calcein Quenching Fluostar Assay |
| Description: | A calcein quenching fluostar assay was performed in order to investigate the biological activity of the newly synthesized examples 1 to 57. This type of assay is disclosed in J. Biol. Chem., 2011, 286, 44319-44325 and Am. J. Physiol. Renal Physiol. (2010), 298, F224-230.The buffers used in the assay were prepared with the following compounds and quantities.500 ml of 4× buffer:3.2 mM MgSO4.7H2O (0.395 g)20 mM KCl (0.746 g)7.2 mM CaCl.2H2O (0.530 g)100 mM NaHepes (13.02 g)pH 7.4 w. HClTetracyclin Stock: Wash buffer (μl) Sucrose buffer (μl) 4x buffer 80000 35000 NaCl (1M) 34080 14910 H2O 199520 18970 Probenecid 6400 2800 Sucrose (1M) 0 68320 Total 320000 140000The total probenecid required to prepare the wash buffer and sucrose buffer is 6400+2800=9200 μl. An additional 500 μl of probenecid (5 plates at 100 μl each) is also required. Therefore, the total probenecid required is 9200 μl+500 μl=9700 μl. Sufficient probenecid is prepared using:690 mg probenecid;4850 μl NaOH 1M;1213 μl 4× buffer; and3638 μl H2O.Assay Experimental Protocol:1) Two days prior to commencement of the assay, seed 10,000 cells/well of 96 well black clear bottom plate (Greiner Poly-lysin plate). A 1:1 mix of Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM: F12) was obtained from Gibco. Tetracycline stock of 5 mg/ml in 96% ethanol is used. Medium: DMEM/F12/10% Donor Bovine Serum, Human AQP9 cell line+1:270,000 tetracyclin, mouse AQP9 cell line+1:2,700,000 tetracycline.2) Day of assay: Flick/slam off the medium and add 50 μl/well of loading solution: 5 ml DMEM/F12/10% Donor Bovine Serum, 25 μl Calcein AM from freshly dissolved aliquot in 50 μl DMSO (VWR #734-1434), and 100 μl Probenecid.3) Incubate the well for 90 minutes at 37° C.4) Perform one wash with 75 μl wash buffer.5) Add 75 μl of an example compound prepared in wash buffer per well.Example compounds are prepared in 500 μl U bottom PP plates (NUNC). 2.7 μl Substance in DMSO are added to row A; 180 μl of wash buffer+1% DMSO are added to rows B H. 90 μl from row A are transferred and mixed with all other wells (up to row G) to make a 3-fold dilution series.6) Assay in FLUOstar Optima at 25° C. Settings buffer addition at 135 μl/seconds, add 75 μl/well, record time course for 30 seconds, add sucrose buffer 3.6 seconds into recording.7) Normalization to initial in Excel.8) Fit to exponential decay function in GraphPad Prism 5.0, then arrange half live shrinking values according to wells and fit dose-response curves. |
| Affinity data for this assay | |
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