Near-Infrared DyLight690 Tracer in vivo Active Caspase (VAD) Assay

1. Prepare samples and controls., 2. Dilute 10X Injection Buffer 1:10 with 45 mL diH20., 3. Reconstitute NIR-FLIVO 690 Free Dye with 50 μL DMSO., 4. Dilute NIR-FLIVO 690 Free Dye 1:10 with 450 μL 1X Injec¬tion Buffer., 5. Inject 50 μL intravenously., 6. Let NIR-FLIVO 690 Free Dye circulate for 4 hours., 7. Image the animal. NIR-FLIVO 690 Free Dye excites at 690 nm and emits at 709 nm., 8. If not viewing directly, excise tissue and process samples for further analysis., 9. Optional: label excised samples with additional stains, such as Hoechst 33342, or an antibody., 10. If desired, fix excised samples., 11. Analyze excised samples with a fluorescence microscope or flow cytometer.