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NIR-FLIVO® 747 Tracer In vivo Assay


FLIVO® (FLuorescence in vIVO) is a powerful method for non-invasive detection of caspase activity in vivo. NIR-FLIVO 747 Tracers are non-cytotoxic, cell-permeant fluorescent inhibitors of caspases optimized for use in whole live animals. ICT’s NIR-FLIVO 747 Tracer probe contains the preferred binding sequence for most caspases (Val-Ala-Asp or VAD). This preferred poly caspase tripeptide binding sequence is labeled with *Dylight® 747 dye and a fluoromethyl ketone (FMK) reactive entity.

FLIVO® is a registered trademark of ICT. *Dylight® is a registered trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.

Catalog # Product Size Quantity Price
9114 20 Tests $396.00
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FLIVO® kits provide a simple and accurate method to detect caspase activity in vivo. Like our FLICA® probes but optimized for whole live animal labeling. To label cells containing active caspases, inject FLIVO intravenously and let it circulate ~60 minutes. Because the reagent is cell-permeant, it readily diffuses in and out of all cells that it encounters as it circulates throughout the body. If there are active caspase enzymes inside a cell, FLIVO will form an irreversible covalent bond with the caspase active site. The bound FLIVO probe will remain inside the cell if the cell membrane is intact. Any unbound FLIVO is removed from the circulation of the animal in about an hour. The remaining near-infrared fluorescent signal in the tissue is a direct measure of caspase activity that occurred at the time the reagent was injected. Once the excess FLIVO has cleared from the body of the animal, the animal is ready for analysis. No further staining is necessary. Apoptotic cells and tissues can be viewed using whole live animal imagers. Alternatively, target tissues can be removed and processed for analysis. FLIVO is very sensitive and will pick up naturally occurring background apoptosis. Apoptotic cells have more active caspases than control cells, therefore they fluoresce brighter with FLIVO.

1. Prepare samples and controls.
2. Dilute 10X Injection Buffer 1:10 with 45 mL diH20.
3. Reconstitute NIR-FLIVO 747 Tracer with 50 μL DMSO.
4. Dilute NIR-FLIVO 1:10 with 450 μL 1X Injection Buffer.
5. Inject 50 μL intravenously.
6. Let NIR-FLIVO circulate for 4 hours.
7. Image the animal. NIR-FLIVO 747 Tracer excites at 747 nm and emits at 776 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.

Apoptotic tissues in murine tumor models were imaged in vivo with NIR-FLIVO® 747 Tracer (*Dylight® 747-VAD-FMK, kit #9114) us¬ing the CRi Maestro™ imaging system. NIR-FLIVO 747 was used to non-invasively monitor the efficacy and time kinetics of an experimental anti-cancer treatment. After receiving a placebo or an experimental treatment, negative control mice (left image) and experimentally treated mice (right image) were injected intravenously with NIR-FLIVO 747 Tracer and imaged non-invasively at various time points with the CRi Maestro™ imaging system. Apoptotic tumor tissues fluoresce bright blue in the experimental group subjects, indicating that the experimental treatment had an apoptotic effect.

NIR 747 Tracer Mice Image

The image shown was acquired using the 747 and 690 FLIVO agents, 48 hr post probe injection, in athymic nude mice xenografts with flank bearing DiFi (a colorectal cancer cell line) tumors. Imaging agent was administered via retro-orbital injection using concentrations recommended by Immunochemistry (7.5nmol). Image and data provide by Matthew R. Hight and H. Charles Manning, Ph. D., Vanderbilt University.

NIR 747 Tracer Colorectal DiFi Xenograft Image

• Reagent Name: NIR-FLIVO® 747 Tracer (747-VAD-FMK)
• Target: Poly Caspase
• Excitation/Emission: 747 nm/776 nm
• Method of Analysis: Use instrumentation designed for non-invasive live animal imaging, alternatively, excised tissues can be analyzed by fluorescence microscope or flow cytometer.
• Types of Samples: Whole animal fluorescence imaging, excised tissues
• Storage: -20°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 9114:
• 2 vials of NIR-FLIVO® 747 Tracer (*Dylight® 747-VAD-FMK), 10 Tests per vial, 77.3 μg per vial, #6308
• 1 bottle of 10X Injection Buffer, 5 mL, #6220
• Kit Manual

FLIVO® is a registered trademark of ICT. *Dylight® is a registered trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.

Wu Q, Berglund AE, Wang D, MacAulay RJ, Mulé JJ, Etame AB. Paradoxical epigenetic regulation of XAF1 mediates plasticity towards adaptive resistance evolution in MGMT-methylated glioblastoma. Sci Rep. 2019 Oct 1;9(1):14072. doi: 10.1038/s41598-019-50489-2. Full Text