Powerful new reagents for near infrared imaging of apoptosis in live animals.
WHAT IS NIR-FLIVO™?
Researchers can now image apoptosis in living animals using ICTs new NIR-FLIVO™ fluorescent in vivo apoptosis detection kits. NIR-FLIVO™ is an injectable fluorescent tracer used to quantitate caspase activity in the cells of live animals. NIR-FLIVO™ is a direct tracer; once labeled, the animal is ready for imaging and no further processing is necessary.
NIR-FLIVO™ is very specific. Only active caspase enzymes will form a covalent bond with the reagent, therefore only cells undergoing apoptosis will fluoresce. If an experimental condition or disease is triggering apoptosis in the animal subject, the increased caspase activation in the affected tissues will result in brighter staining compared with a control. The signal intensity may be quantified in vivo with an optical imaging system or ex vivo by analyzing tissue samples with a flow cytometer. With NIR-FLIVO™, the animals response to treatment or disease may be determined quickly, allowing for the conditions to be adjusted accordingly. It may also be used to investigate apoptotic side effects of treatment or disease and to calculate dose response.
HOW IS NIR-FLIVO™ USED?
NIR-FLIVO™ is very easy to use. Just inject it into the animal intravenously and let it circulate 30 minutes to several hours. Apoptotic cells will fluoresce and can be counter-stained with other reagents (such as Nissl red). Animals may be analyzed using a whole animal imaging system. NIR-FLIVO™-stained tissues may be viewed directly through a window chamber system or other accessible cavity, or excised cells may be analyzed ex vivo after sacrifice or biopsy with a fluorescence microscope, plate reader, or flow cytometer. Tissues may be fixed or frozen for future analysis; protect from light during handling.
WHY DOES NIR-FLIVO™ WORK?
Because NIR-FLIVO™ is cell-permeant, it diffuses in and out of all cells as it circulates throughout the body. If there is an active caspase enzyme inside a cell, it will form an irreversible covalent bond with NIR-FLIVO™ and retain the fluorescent signal within the cell indefinitely.
Unbound FLIVO™ is rapidly cleared from the circulation of the animal, although it may be held in the liver for 3-4 hours, and it may accumulate in the bladder and the feces until excreted. NIR-FLIVO™ is very sensitive and will even pick up naturally occurring background apoptosis.
WHAT WAVELENGTHS DOES NIR-FLIVO™ USE?
NIR-FLIVO™ is available with a 690 or a 747 label. The 690 in vivo NIR-FLIVO™ tracer (NIR-FLIVO™-DyLight®-690-VAD-FMK) excites at 690 nm and emits at 709 nm. The 747 in vivo NIR-FLIVO™ tracer (NIR-FLIVO™-DyLight®-747-VAD-FMK) excites at 747 nm and emits at 776 nm. Use NIR-FLIVO™ alongside labeled antibodies and other probes for dual-staining studies ex vivo.
HOW MANY ANIMALS CAN BE TESTED WITH NIR-FLIVO™?
NIR-FLIVO™ kits can test approximately 20 animals. Each kit contains 2 vials of the tracer and injection buffer. Based on our initial mouse models, each vial contains enough reagent to inject 10 mice or 2 young rats at approximately 300 nanomoles/Kg (please inquire if using other species or larger animals). At the recommended dose, 1 vial treats 10 mice; however, depending on the experimental conditions, more or less may be used.
WHAT ANIMALS AND TISSUES WILL WORK WITH NIR-FLIVO™?
NIR-FLIVO™ will detect apoptosis in all tissues throughout the animal; it is not limited to detecting apoptosis in transfected tumor cells.NIR-FLIVO™ crosses the blood-brain barrier and may be used to quantify neuronal cell death in living animals. FLIVO™ has been successfully used to assess in vivo caspase activity in a variety of cell and tissue types in mice, rats, hamsters, and birds. Set up an initial reagent titration experiment to determine the amount of reagent that will work best for the size of your animal and tissue type.
DOES NIR-FLIVO™ PHOTO-BLEACH?
NIR-FLIVO™ is very resistant to photo-bleaching.
HOW STABLE ARE THE NIR-FLIVO™ STOCK SOLUTIONS?
The aqueous diluted injection buffer should be used within 4 hours. The reconstituted stock in DMSO can be divided into aliquots and froze at -20°C (-80°C is preferred) for up to 6 months.
HOW LONG AFTER INJECTION CAN NIR-FLIVO™ BE OBSERVED?
Some fluorescence has been observed in the tumor up to 22 hours post injection, but after 28 hours, no NIR-FLIVO™ signal was detected.
DOES NIR-FLIVO™ AFFECT A DRUGS IMPACT ON APOPTOSIS?
No, it seems to have no effect on halting the apoptotic process once it has began.
WHAT IS THE TYPICAL DOSING OF NIR-FLIVO™ IN TUMOR MODELS?
Recommended dosing is 300 nmol/kg; however, other models have achieved desirable results using as little as 30 nmol/kg.
HOW LONG MUST I WAIT WHEN IMAGING THE BRAIN?
You can image as soon as 30 min, but we recommend 1 hour.
HOW LONG MUST I WAIT TO IMAGE THE LIVER?
You can image the liver after 4 hours, which is the time required for all non-specific FLIVO™ to clear the liver. If time constraints are present, the liver could possibly be imaged after 3 hours.
ARE THERE ANY ORGANS WHERE NIR-FLIVO™ MIGHT BUILD UP?
NIR-FLIVO™ does not build up, but rather some NIR-FLIVO™ gets cleared through the liver and passes through the intestines before leaving in the animal’s feces. The remaining NIR-FLIVO™ is excreted by the kidney into the urine, so depending on when the animal last relieved itself, there might be significant NIR-FLIVO™ in the bladder. The presence of NIR-FLIVO™ in these organs depends on the amount of time that has elapsed since the injection, the amount of water and food consumed, and the activity level of the animal. (Caution: Be sure the animal does not walk around in the fluorescent urine, otherwise the feet and underside will fluoresce when imaging)
ARE THERE OTHER VARIABLES THAT MIGHT ADD BACKGROUND FLUORESCENCE WHICH SHOULD BE CONSIDERED WHEN USING NIR-FLIVO™?
Be aware of what is in the animal’s feed. Some vegetable matter and grasses can contribute unwanted background fluorescence.
WHAT METHODS OF INJECTING NIR-FLIVO™ IN MICE WORK?
IV injections in both the tail vein and supraorbital vein have shown great results. However, injecting into the peritoneal cavity did not produce good results.
1. Dilute injection buffer with 45 mL diH2O.
2. Reconstitute FLIVO™ with 50 μL DMSO.
3. Dilute FLIVO™ with 450 μL 1X injection buffer.
4. Inject 50 μL intravenously.
5. Let circulate 4 hours.
6. Image the animal.
7. If not imaging in vivo, prepare samples for ex vivo analysis.
8. If desired, label histology samples with additional stains, such as DAPI or an antibody, and/or fix or embed samples.
9. Analyze histology samples with a fluorescence microscope or flow cytometer.
Download your NIR-FLOVO™ brochure (pdf, 374 kb).
NIR-FLIVO™-690 IN VIVO APOPTOSIS KIT
NIR-FLIVO™-690 tracer and injection buffer
20 tests, cat# ICT-9112
NIR-FLIVO™-690 FREE DYE CONTROL KIT
NIR-FLIVO™-690 control and injection buffer
20 tests, cat# ICT-9113
NIR-FLIVO™-747 IN VIVO APOPTOSIS KIT
NIR-FLIVO™-747 tracer and injection buffer
20 tests, cat# ICT-9114
NIR-FLIVO™-747 FREE DYE CONTROL KIT
NIR-FLIVO™-747 control and injection buffer
20 tests, cat# ICT-9115
NIR-FLIVO™ in vivo Apoptosis Kits