Advanced Calcein AM Cell Viability Kit

1. Prepare samples and controls in 0.5 mL 1X Assay Buffer or buffer of choice at a cell density between 3-5 x 105 cells/mL. If samples were cultured in serum-containing medium, wash the samples prior to adding the DAF-2DA dye. 2. Prepare DAF-2DA staining solution by adding 56 μL DAF-2DA to 444 μL water. This is sufficient volume to stain 50 x 0.5 mL or 100 x 0.25 mL samples. 3. Pre-load samples with DAF-2DA staining solution by adding 10 μL into 490 μL cultured cells, or 5 μL into 245 μL cultured cells. 4. Incubate 1 hour at 37°C. 5. Wash samples at least once with 1X Assay Buffer to remove excess dye, and then resuspend in 1X Assay Buffer. 6. Treat cells with test compounds for desired period of time to induce NOS production. Keep cells protected from light. 7. Analyze with a flow cytometer, fluorescence plate reader, or fluorescence microscope. DAF-2DA excites at 488 nm and emits at 515 nm.