ICT’s near-infrared (NIR)-FLIVO® Tracers are used in conjunction with NIR-FLIVO Free Dye Control Assays. The NIR-FLIVO Free Dye Control Assay uses the NIR-FLIVO Free Dye Control reagent (*Dylight® 690 Free Dye). When injected, both the Free Dye Control and the Tracer reagents will generate a fluorescent signal, but only the Tracer should bind to active caspases and remain inside an apoptotic cell, while signal associated with the Free Dye Control represents background noise. FLIVO® is a registered trademark of ICT. *Dylight® is a registered trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
- Prepare samples and controls.
- Dilute 10X Injection Buffer 1:10 with 45 mL diH20.
- Reconstitute NIR-FLIVO 690 Free Dye with 50 μL DMSO.
- Dilute NIR-FLIVO 690 Free Dye 1:10 with 450 μL 1X Injec¬tion Buffer.
- Inject 50 μL intravenously.
- Let NIR-FLIVO 690 Free Dye circulate for 4 hours.
- Image the animal. NIR-FLIVO 690 Free Dye excites at 690 nm and emits at 709 nm.
- If not viewing directly, excise tissue and process samples for further analysis.
- Optional: label excised samples with additional stains, such as Hoechst 33342, or an antibody.
- If desired, fix excised samples.
- Analyze excised samples with a fluorescence microscope or flow cytometer.
FLIVO® is a registered trademark of ICT. *Dylight® is a registered trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.