| Assay Method Information | |
| | Influenza Antiviral Assay |
| Description: | Human lung carcinoma A549 cells (ATCC, Manassas, Va.) were plated at a density of 5×104 cells/mL (10×103 cells/well) in assay media (Ham's F12 media supplemented with 0.3% FBS, 1% penicillin/streptomycin, 1% L-Glutamine, and 1% non-essential amino acids (all Mediatech, Manassas, Va.) and 1% DMSO (Sigma-Aldrich, St Louis, Mo.)) in white 96-well plates. Cells were infected with 250 IU/well of Influenza strain A549 A/WSN/33 (H1N1) (Virapur, San Diego Calif.) and incubated for 20 hours at 37° C., 5% CO2. The cell culture supernatant was aspirated off and 50 μL of 25 μM 2′-(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid (Sigma-Aldrich) dissolved in 33 mM MES, pH 6.5 (Emerald Biosystems, Bainbridge Island, WA) was added to the cells. After incubation for 45 min at 37° C., reactions were stopped by addition of 150 μL stop solution (100 mM glycine, pH 10.5, 25% ethanol, all Sigma-Aldrich). Fluorescence was measured with excitation and emission filters of 355 and 460 nm, respectively, on a Victor X3 multi-label plate reader (Perkin Elmer, Waltham, Mass.). Cytotoxicity of uninfected parallel cultures was determined by addition of 100 μL of CellTiter-Glo reagent (Promega, Madison, Wis.), and incubation for 10 min at RT. Luminescence was measured on a Victor X3 multi-label plate reader. |
| Affinity data for this assay | |
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