Nitric Oxide Synthase Assay

1. Prepare samples and controls in fresh cell culture medium (alternative buffers such as PBS may be used) . 2. Reconstitute Autophagy Probe, Red with 100 μL DMSO. 3. Dilute Autophagy Probe, Red 1:5 by adding 400 μL diH2O, PBS, or fresh cell culture medium. 4. Add diluted Autophagy Probe, Red to each sample at 1:50 spike (e.g. spike at 1:50 by adding 10 μL to 490 μL sample) . The ideal staining concentration can vary based on cell line, application, etc. and should be determined by the end user. The recommended sample size is 0.5 mL. 5. Incubate approximately 1 hour. 6. Dilute 10X Cellular Assay Buffer 1:10 with diH2O. 7. Remove media and wash cells 3 times: add 1X Cellular Assay Buffer or fresh cell culture medium and spin cells. 8. Resuspend cell pellet in 1X Cellular Assay Buffer (typically 0.5 mL per sample) . 9. Analyze with a flow cytometer (using a green/yellow laser and appropriate filter) . Autophagy Probe, Red excites at 590 nm and emits at 620 nm.