NIR-FLIVO™ 747 in vivo Apoptosis Tracer

NIR FLIVO™ 747 in vivo Apoptosis Tracer: 10 test vial, cat. # 6308
NIR FLIVO™ 747 in vivo Apoptosis Kit: 20 test kit, cat. # 9114

NIR FLIVO™ in vivo Apoptosis Tracers and Kits provide an accurate and direct method to detect in vivo apoptosis for small animal imaging. 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, FLIVO™ tracers circulate throughout the body and preferentially form covalent bonds with active caspases, marking apoptotic cells as fluorescent. Bound FLIVO™ tracer will remain inside the cell as long as the cell membrane is intact. Any unbound FLIVO™ is removed with the natural circulation of the animal. The resulting fluorescent signal within samples is a direct measure of apoptosis that was occurring at the time the reagent was injected.

FLIVO™ is non-toxic, cell-permeant, and crosses the blood-brain barrier. Reagent titration experiments should be performed to determine the amount of reagent that will work best for the size of the animal and the target tissue type. FLIVO™ is a direct stain and eliminates false positives that may arise from ex vivo manipulation of tissue. This gives a true representation of the induction of apoptosis as a result of the experimental condition.

Applications:
FLIVO™ may be used in animal models to monitor the efficacy of treatment or the effects of disease. Among its demonstrated uses, it has been used successfully in murine models of cancer, rat brain studies, and avian brain studies.  NIR-FLIVO in vivo apoptosis tracers, which utilize near-infrared fluorescent DyLight® labels, may be imaged non-invasively with small animal imaging systems such as the CRi Maestro and the Caliper IVIS.

Related Reagents Available:

• NIR-FLIVO™ 690 Free Dye Control Reagent and Kit
  containing DyLight® 690 Carboxylic Acid Form Free Dye
• NIR-FLIVO™ 747 Free Dye Control Reagent and Kit
  containing DyLight® 747 Carboxylic Acid Form Free Dye
• 10X Injection Buffer
  for use with FLIVO Tracers and Controls,15 mL bottle, #6220

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™ in vivo apoptosis imaging tracers to assess levels of apoptosis in live animals. Sample Protocols for NIR-FLIVO™ 690:

Preparation and Injection:

1. Determine time points at which to investigate the level of apoptosis. NIR-FLIVO will detect apoptosis that is occurring at the time of injection.
2. Establish positive and negative controls, and expose animals to experimental condition.
3. Reconstitute NIR-FLIVO™ with 50mcL DMSO to form the stock solution (which can be frozen for future use).
4. Dilute the 10x Injection Buffer 1:10 with diH2O and sterilize by filtration.
5. Add 450mcL 1X injection buffer to the reconstituted NIR-FLIVO™ stock solution.
6. Inject 50mcL of diluted NIR-FLIVO™ intravenously. Note: diluted 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.
3. The same animal subject should be able to be reinjected and reanalyzed with NIR-FLIVO within 1-4 days, depending on the rate of cellular apoptosis.

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, 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-labeled samples with a fluorescent microscope, plate reader, or optical imaging system equipped with the proper laser and filter pairing.

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, 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-labeled samples with a fluorescent microscope, plate reader, or optical imaging system equipped with the proper laser and filter pairing.

FLIVO™ CITATIONS

1. Griffin, R.J. et al. Use of a fluorescently labeled poly-caspase inhibitor for in vivo detection of apoptosis related to vascular-targeting agent arsenic trioxide for cancer therapy. Technology in Cancer Research and Treatment. 6, 651-654 (2007).
2. Tsai, Y.C. et al. The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nat Med. 13, 1504-1509 (2007).
3. Cursio, R., Colosetti, P., Auberger, P., and Gugenheim, J. Liver apoptosis following normothermic ischemia-reperfusion: in vivo evaluation of caspase activity by FLIVO™ assay in rats. Transplant. 40, 2038–2041 (2008).
4. Gobeil, S., Zhu, X., Doillon, C.J., and Green, M.R. A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene. Genes Dev. 22: 2932-40 (Nov. 2008).
5. Lee, B.W., Olin, M.R., Johnson, G.L., and Griffi n, R.J. In vitro and in vivo apoptosis detection using membrane permeant fl uorescent-labeled inhibitors of caspases. Methods Mol Biol. 414:109-35 (2008).
6. Cursio, R., et al. Tyrosine phosphorylation of insulin receptor substrates during ischemia/ reperfusion-induced apoptosis in rat liver. Langenbecks Arch Surg. 394, 123-131 (2009).
7. Delgado-Martín, C., et al. A protocol to detect apoptotic dendritic cells in murine lymph nodes using multiphoton microscopy. Nature Protocols. DOI: 10.1038/nprot. 2009.133.
8. Erman, A., Zupancic, D., and Jezernik, K. Apoptosis and desquamation of urothelial cells in tissue remodeling during rat postnatal development. J Histochem Cytochem. 57, 721-730 (August 2009).
9. Escribano, C, et al. CCR7-Dependent Stimulation of Survival in Dendritic Cells Involves Inhibition of GSK3. J Immunol. 183: 6282-6295 (2009).
10. Medina, M.A., Nguyen, J.T., McCormack, M.M., Randolph, M.A., and Austen, W.G. A high-throughput model for fat graft assessment. Laser Surg Med. 41:738-744 (2009).
11. Riol-Blanco, L., et al. Immunological synapse formation inhibits, via NF-kappaB and FOXO1, the apoptosis of dendritic cells. Nat Immunol. 10(7), 753-760 (2009).
12. Van der Most, R.G., et al. Cyclophosphamide chemotherapy sensitizes tumor cells to TRAIL-dependent CD8 T cell-mediated immune attack resulting in suppression of tumor growth. PLos ONE 4:e6982 (September 2009).
13. Slatter, T.L., Ganesan, P., Holzhauer, C., Mehta, R., Rubio, C., Williams, G., Wilson, M., Royds, J.A., Baird, M.A., and Braithwaite, A.W. p53-mediated apoptosis prevents the accumulation of progenitor B cells and B-cell tumors. Cell Death Diff. 17:540-550 (2010).
14. Perrone, L., Devi, T.S., Hosoya, K-I., Terasaki, T., and Singh, L.P. Inhibition of TXNIP expression in vivo blocks early pathologies of diabetic retinopathy. Cell Death Dis. 1, e65; doi:10.1038/cddis.2010.42 (2010). 
15. Olin, M., Roy, S., and Molitor, T. In Vivo Morphine Treatment Synergistically Increases LPS-Induced Caspase Activity in Immune Organs. J Neuroimmune Pharmacol. 5, 4:546-552, DOI: 10.1007/s11481-010-9209-8 (2010).
16. Cursio, R, et al. 2010. Induction of Different Types of Cell Death After Normothermic Liver Ischemia-Reperfusion. Transplant P. 42, 3977-3980.
17. Altmeyer, A., et al. Cell Death After High-LET Irradiation in orthotopic human hepatocellular carcinoma in vivo. In Vivo. 25: 1-9 (2011).
18. Schiavoni, G., et al. Cyclophosphamide Synergizes with Type I Interferons through Systemic Dendritic Cell Reactivation and Induction of Immunogenic Tumor Apoptosis. Cancer Res. 71: 768-778 (2011). 
19. Ju, T.C., et al. Nuclear translocation of AMPK-α1 potentiates striatal neurodegeneration in Huntington’s disease. J Cell Biol. 2011 Jul 18;194(2):209-27. Doi: 10.1083/jcb.201105010 (2011). [Abstract]
20. Darzynkiewicz, Z., Pozarowski, P., Lee, B.W., and Johnson, G.L. Fluorochrome-Labeled Inhibitors of Caspases: Convenient In Vitro and In Vivo Markers of Apoptotic Cells for Cytometric Analysis. Methods Mol Biol, 1, DNA Damage Detection In Situ, Ex Vivo, and In Vivo. 682:103-14 (2011).
21. Merrick, B., Dhungana, S., Williams, J., Aloor, J., Peddada, S., Tomer, K., Fessler, M. Proteomic Profiling of S-acylated Macrophage Proteins Identifies a Role for Palmitoylation in Mitochondrial Targeting of Phospholipid Scramblase 3. Mol Cell Proteomics. 10:M110.006007 (2011). [Abstract]
22. Delgado-Martín, C., Escribano, C., Pablos, J. L., Riol-Blanco, L., Rodríguez-Fernández, J. L. Chemokine CXCL12 Uses CXCR4 and a Signaling Core Formed by Bifunctional Akt, Extracellular Signal-regulated Kinase (ERK)1/2, and Mammalian Target of Rapamycin Complex 1 (mTORC1) Proteins to Control Chemotaxis and Survival Simultaneously in Mature Dendritic Cells. J Biol Chem. 286:37222-37236 (2011). [Abstract] 

Our Newest FLIVO Citations describe the use of the green fluorescence FAM-FLIVO tracer for visualizing apoptotic hepatocytes via endomicroscopy:

In vivo real-time imaging of the liver with confocal endomicroscopy permits visualization of the temporospatial patterns of hepatocyte apoptosis
Martin Goetz, Jacqueline V. Ansems, Peter R. Galle, Marcus Schuchmann, and Ralf Kiesslich
Am J Physiol Gastrointest Liver Physiol. 2011; 301:G764-G772.  [Abstract]

Confocal laser endomicroscopy in dynamic evaluation of hepatic apoptosis in vivo
Fanyin Meng and Gianfranco Alpini
Am J Physiol Gastrointest Liver Physiol. 2011; 301:G762-G763.  [Full Text]

How does FLIVO™ work?
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, FLIVO™ 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™ probe 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.

What is the fluorescent detection label on NIR-FLIVO?
Our first release of NIR-labeled FLIVO tracers utilize DyLight^® fluorophores in the near-infrared range for noninvasive imaging with whole animal imaging systems.

How may FLIVO be detected?
Our new NIR-FLIVO™ in vivo apoptosis tracers may be imaged with whole animal optical imaging systems, such as the Caliper™/Xenogen IVIS^® machine and the CRi Maestro™.  NIR-FLIVO™ 690 in vivo apoptosis tracer has an optimal emission of 709 nm, and NIR-FLIVO™ 747 in vivo apoptosis tracer has 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. The red SR fluorophore on our SR-FLIVO tracer will penetrate up to 3 mm of skin tissue for detection with IVIS^®; this works best on hairless animals with light skin. ICT is developing additional tracers for non-invasive optical imaging as well as tracers for alternative detection methods.

How many tests can be run with the kit?
Our NIR-FLIVO kits include two vials of the NIR-FLIVO in vivo apoptosis tracer, each containing enough reagent for 10 injections with small animals.  The necessary amount of FLIVO per animal will vary by experiment, depending on the size of the animal and the expected level of apoptosis. We recommend that the investigator perform titration experiments to optimize the necessary amount for their study. Volume sales of the NIR-FLIVO tracers are available; please inquire for a quote.

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.