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NIR-FLIVO® 690 Tracer In vivo Assay


FLIVO® (FLuorescence in vIVO) is a powerful method for non-invasive detection of caspase activity in vivo. NIR-FLIVO 690 Tracers are non-cytotoxic, cell-permeant fluorescent inhibitors of caspases optimized for use in whole live animals. ICT’s NIR-FLIVO 690 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® 690 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
9112 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 690 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 690 Tracer 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.

Brain abscesses were induced in mice following the intracerebral inoculation of live Staphylococcus aureus. Animals received intravenous injections of NIR-FLIVO 690 Tracer (kit #9112) or NIR-FLIVO 690 Free Dye Control (kit #9113) at 17 hours post-infection, whereupon signals were acquired 1 hour later from brain tissues ex vivo using an IVIS® Spectrum™ (Caliper Life Sciences). Strong caspase activity was associated with brain abscesses following administration of the NIR-FLIVO 690 Tracer (right image), whereas minimal signal was detected in animals injected with the NIR-FLIVO 690 Free Dye Control (left image).
690 Tracer and Free Dye data

Here are whole animal images using FLIVO to study brain infections. Image take with a Carestream In Vivo FX PRO imager. Adult wild-type Balb/c mice were either given a control (#1&2 top and middle) or intranasal inoculation with the HSV-1 virus (#3 bottom), which is absorbed thru the epithelium and is known to induce apoptosis in the brain. 7 days later, at the peak of infection, the mice were injected IV in the tail vein without any reagent (#1) or with NIR-690 (#2 and #3 middle and bottom), and imaged 4 hours later. We can see strong caspase activity located in the brain of the bottom animal treated with HSV-1 and injected with NIR690 (#3 bottom). Minimal signal was detected in the liver region of both mice injected with NIR690 (#2 and #3) as the liver is the route of clearance for FLIVO tracers. There is no signal in the uninfected control (#1 top), therefore #2 (middle) reveals background staining.

NIR 690 Tracer Mouse Brain Image

• Reagent Name: NIR-FLIVO® 690 Tracer (690-VAD-FMK)
• Target: Poly Caspase
• Excitation/Emission: 690 nm/709 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 9112:
• 2 vials of NIR-FLIVO® 690 Tracer (*Dylight® 690-VAD-FMK), 10 Tests per vial, 71.4 μg per vial, #6306
• 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.