NIR-FLIVO™ 747 Free Dye Control (DyLight® 747 Carboxylic Acid Form)

NIR-FLIVO™ 747 Free Dye Control is offered for use alongside the NIR-FLIVO™ 747 in vivo Apoptosis Tracer.  This imaging control reagent is offered as individual reagent vials (catalog #6309) or in a kit (catalog #9115).

NIR-FLIVO™ 747 Free Dye Control Reagent (#6309):
Synonyms: NIR-FLIVO™ 747 Control , DyLight® 747 (Carboxylic Acid Form)
Size: 10 tests; 53.6 µg per vial
Molecular weight: 712.96

NIR-FLIVO™ 747 Free Dye Control Kit (#9115) contains:

• NIR-FLIVO™ 747 Free Dye Control Reagent #6309, 2 vials
• 10x injection buffer (5 ml) #6220, 1 bottle 

NIR FLIVO™ in vivo Apoptosis Tracers and Kits provide an accurate and direct method to image apoptosis in vivo. FLIVO™ (FLuorescence in vIVO) tracers are non-cytotoxic fluorescent inhibitors of the class of cysteine proteases known as caspases, which are associated with the execution of apoptosis. After intravenous injection, nontoxic FLIVO™ in vivo apoptosis tracers will illuminate caspase-positive, apoptotic cells and tissues in living animal models.

NIR-FLIVO™ in vivo apoptosis tracers may be imaged non-invasively with whole animal imaging systems such as the CRi Maestro and the Caliper IVIS.  The FLIVO™ method of in vivo apoptosis detection eliminates false positives associated with ex vivo staining protocols, giving a true representation of the induction of apoptosis as a result of the experimental condition.

Label, Excitation, and Emission of FLIVO™ Tracers and Controls:
NIR-FLIVO™ 690 Tracer and Control: DyLight® 690; 690 nm/ 709 nm
NIR-FLIVO™ 747 Tracer and Control: DyLight® 747; 747 nm/ 776 nm
FAM- FLIVO™ Tracer: carboxyfluorescein; 492 nm/ 520 nm
SR-FLIVO™ Tracer: sulforhodamine B; 565 nm/ 600 nm

NIR-FLIVO™ and FLIVO™ are trademarks of ImmunoChemistry Technologies, LLC.  DyLight® is a registered trademark or Thermo Fisher Scientific, Inc. and its subsidiaries. CRi and Maestro™ are trademarks of Cambridge Research & Instrumentation, Inc. IVIS® is a registered trademark of Caliper Life Sciences, Inc.

Sample Protocol:

ICT offers a growing range of novel tools for in vivo apoptosis detection. Use our NIR-FLIVO™ Free Dye Control reagents to run controls alongside our NIR-FLIVO™ in vivo apoptosis imaging tracers. Sample Protocols for the NIR-FLIVO™ 747 Free Dye Control Reagent and Kit:

Preparation and Injection:

1. Establish positive and negative controls, and expose animals to experimental condition.
2. Reconstitute NIR-FLIVO™ Control with 50mcL DMSO to form the stock solution (which can be frozen for future use).
3. Dilute the 10x Injection Buffer 1:10 with diH2O and sterilize by filtration.
4. Add 450mcL 1X injection buffer to the reconstituted NIR-FLIVO™ Control stock solution.
5. Inject 50mcL of diluted NIR-FLIVO™ 747 Control intravenously. Note: diluted NIR-FLIVO must be used within 8 hours. 

Analysis by in vivo Imaging:

1. Allow reagent to circulate 4 hours before imaging.
2. Image with a whole animal imaging system for NIR fluorescence.

Analysis of Non-hepatic Histological Samples ex vivo:

1. Allow reagent to circulate in vivo 30-60 minutes before preparing samples (i.e. biopsies, thin tissue sections, etc).
2. Protect samples from exposure to light.
3. If desired, fix with paraformaldehyde-based fixatives, snap-freeze, and/or disassociate tissue into cell suspensions.
4. If desired, label samples with an additional stain, such as DAPI or an antibody.
5. Analyze NIR-FLIVO™ 747 Control-labeled samples with a fluorescent microscope or optical imaging system equipped with the proper lasers and filters.

Analysis of Hepatic Histological Samples ex vivo:

1. Allow reagent to circulate in vivo 4 hours before preparing samples (i.e. biopsies, thin tissue sections, etc).
2. Protect samples from exposure to light.
3. If desired, fix with paraformaldehyde-based fixatives, snap-freeze, and/or disassociate tissue into cell suspensions.
4. If desired, label samples with an additional stain, such as DAPI or an antibody.
5. Analyze NIR-FLIVO™ 747 Control-labeled samples with a fluorescent microscope or optical imaging system equipped with the proper lasers and filters.

Minimal signal from NIR-FLIVO™ 747 Free Dye Control (DyLight® 747 carboxylic acid form) was detected in pancreatic tumor xenografts (Figures 3 and 4) using the CRi Maestro™ imaging system (Caliper Life Sciences).

NIR-FLIVO™ 747 in vivo apoptosis tracer was used to measure caspase activity and image apoptosis in BALB/c NU/NU mice bearing DU-145 pancreatic tumor xenografts (circled in Figure 1). Mice were injected with NIR-FLIVO™ 747 in vivo Apoptosis Tracer (Figures 1 and 2) or the NIR-FLIVO™ 747 Free Dye as an imaging control (Figures 3 and 4) and imaged non-invasively.  Mice injected with the NIR-FLIVO™ 747 Tracer show a greater NIR signal in treated tumor areas than the mice that received the injection of NIR-FLIVO™ 747 Free Dye Control Reagent.

Data provided courtesy of collaborators at the Uni­versity of Illinois College of Medicine, Rockford.

FLIVO™ is a trademark of ImmunoChemistry Technologies, LLC. DyLight® is a registered trademark of Thermo Fisher Scientific, Inc. and its subsidiaries. CRi and Maestro™ are trademarks of Cambridge Research & Instrumentation, Inc.

What is the fluorescent detection label on the NIR-FLIVO™ products?
Our first release of NIR-labeled FLIVO™ tracers utilize DyLight^® fluorophores in the near-infrared range for noninvasive imaging with whole animal imaging systems. We offer the carboxylic acid form of these NIR free dyes for running imaging controls alongside the apoptosis tracers.

How may FLIVO™ be detected?
Our new NIR-FLIVO™ in vivo apoptosis tracers and controls may be imaged with whole animal optical imaging systems, such as the Caliper™/Xenogen IVIS^® machine and the CRi Maestro™.  NIR-FLIVO™ 690  Free Dye Control and NIR-FLIVO™ 690 in vivo apoptosis tracer have an optimal emission of 709 nm.  NIR-FLIVO™ 747 Free Dye Control and NIR-FLIVO™ 747 in vivo apoptosis tracer have an optimal emission of 776 nm.

Our first-generation FLIVO™ tracers (FAM,SR) are compatible with in vivo labeling and ex vivo analysis procedures, yet have limited noninvasive imaging characteristics. ICT is developing additional tracers for non-invasive optical imaging as well as probes for alternative detection methods.

How many tests can be run with the vials and the kit?
Our NIR-FLIVO™ Free Dye Control Kits include two vials of the NIR-FLIVO Free Dye Control Reagent, which is the carboxylic acid form of the DyLight^® free dye.  Each vial contains enough reagent for 10 injections with small animals. Volume sales of the NIR-FLIVO controls are available; please inquire for a quote.

How do FLIVO™ in vivo apoptosis tracers detect apoptosis?
After intravenous injection, FLIVO readily diffuses in and out of all cells as it circulates throughout the body. If there are active caspase enzymes inside a cell, the FLIVO™ apoptosis tracer will form an irreversible covalent bond with a reactive cysteine on the large subunit of the caspase heterodimer, thereby inhibiting further enzymatic activity and keeping the FLIVO signal inside the cell. The bound FLIVO™ tracer will remain inside the cell as long as the cell membrane is intact. Any unbound FLIVO™ is removed from the circulation of the animal in about an hour. The resulting fluorescent signal within samples is a direct measure of apoptosis that occurred at the time the reagent was injected.

Call 1-800-829-3194 for technical assistance or email Technical Support: help {at} immunochemistry.com.

NIR-FLIVO™ and FLIVO™ are trademarks of ImmunoChemistry Technologies, LLC.  DyLight® is a registered trademark or Thermo Fisher Scientific, Inc. and its subsidiaries. CRi and Maestro™ are trademarks of Cambridge Research & Instrumentation, Inc. Xenogen® and IVIS® are registered trademarks of Caliper Life Sciences, Inc.