Red SR FLIVO™ in vivo Apoptosis Kit

FLIVO™ (FLuorescence in vIVO) in vivo apoptosis tracers are non-cytotoxic fluorescent inhibitors of the class of cysteine proteases known as caspases, which are associated with the execution of apoptosis. ICT's FLIVO™ tracers preferentially form covalent bonds with active caspases, causing cells undergoing apoptosis to fluoresce. SR-FLIVO™ (SR-VAD-FMK), our red fluorescent in vivo apoptosis probe, is a sulforhodamine-B (SR) conjugate of valylalanylaspartic acid (VAD) fluoromethyl ketone (FMK). FLIVO™ is also available with a green fluorescent label (FAM) and two near-infrared (NIR) fluorescent labels. See the FLIVO page for more information.

Simple and Most Accurate Method of in vivo Apoptosis Detection
To label apoptotic cells, inject FLIVO intravenously and let it circulate ~60 minutes. The cell-permeant reagent will diffuse through all cells as it circulates throughout the body. Upon encountering active caspases, 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 labeling its location. 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 remaining green or red fluorescent signal in the tissue is a direct measure of caspase activity that occurred at the time the reagent was injected.

Eliminate False Positives
Once the animals have been injected with FLIVO™ and the unbound reagent has been allowed to clear from the non-apoptotic tissues, the tissues are ready for analysis and no further staining is necessary. FLIVO™ eliminates any false positives that may otherwise arise from post-sacrifice tissue manipulation. This gives a true representation of the induction of apoptosis in vivo as a result of the experimental condition.

Fluorescence Analysis Methods
Thin tissue sections may be prepared after sacrificing the animal. Tissues labeled with FLIVO™ can be counter-stained with other reagents, such as Nissl, DAPI, or DRAQ5, and fixed or frozen for future analysis. The fluorescence intensity can be quantified by excising the tissue and analyzing cells with a fluorescence plate reader or a flow cytometer with a green excitation laser. SR-FLIVO™ excites at 565 nm and emits at 600 nm.

Future Clinical Applications
ICT is further developing FLIVO (research) and related products as diagnostic detection methods to more accurately assess tumor shrinkage, neurodegeneration, retinal degeneration, and other degenerative conditions in many animal models and cell and tissue types. Every day, we are finding new ways to use our reagents to develop cures, better manage disease, and personalize treatments. One day, these reagents will be used in clinical labs to tell doctors and their patients if their condition is improving.

Collaborate with ICT
If you need an in vivo probe for your research, have a promising tracer technology, or would like to collaborate with ICT, please contact Gary L. Johnson or Dr. Brian W. Lee at 1-800-829-3194 / 952-888-8788.

Ordering
To buy SR-FLIVO, select your desired size from the drop-down menu above, and click on the green "Add to Cart" button.

Catalog no. 982: SR-FLIVO™ in vivo Apoptosis Kit, red fluorescence, small; $179 USD
Catalog no. 983: SR-FLIVO™ in vivo Apoptosis Kit, red fluorescence, large; $499 USD

Sample Protocol:
ICT offers a growing range of novel tools for in vivo apoptosis detection. Use our fluorescent in vivo apoptosis probes, FLIVO™, to assess levels of apoptosis in live animals. Sample Protocol using Mouse Models:

1. Expose animals to experimental condition, and create positive and negative controls.
2. Reconstitute the reagent with 50mcL DMSO to form the stock concentrate (which can be frozen for future use).
3. Dilute the injection buffer 1:10 with diH2O and sterilize by filtration.
4. Add 550mcL 1X injection buffer to the reagent.
5. Inject ~100mcL of diluted reagent into each mouse.
6. Let circulate 30-60 minutes.
7. Examine tissues under a fluorescent microscope, or sacrifice and excise cells.
8. If desired, label cells with an additional stain, fix, embed, or freeze cells.
9. Analyze cells with a fluorescent microscope, plate reader or flow cytometer.

1. Tsai, YC, et al. 2007. The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nature Medicine. 13, 1504-1509.
2. Griffin, RJ, et al. 2007. 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).
3. Cursio, R, et al. 2008. Liver Apoptosis Following Normothermic Ischemia-Reperfusion: In Vivo Evaluation of Caspase Activity by FLIVO Assay in Rats. Transplant P. 40, 2038-2041.
4. Cursio, R, et al. 2009. Tyrosine phosphorylation of insulin receptor substrates during ischemia/reperfusion-induced apoptosis in rat liver. Langenbecks Arch Surg. 394, 123-131.
5. Delgado-Martín, C, et al. 2009. A protocol to detect apoptotic dendritic cells in murine lymph nodes using multiphoton microscopy. Nature Protocols. DOI: 10.1038/nprot.2009.133. http://www.natureprotocols.com/2009/06/10/a_protocol_to_detect_apoptotic.php
6. Riol-Blanco, L, et al. 2009. Immunological synapse formation inhibits, via NF-kappaB and FOXO1, the apoptosis of dendritic cells. Nat Immunol, 10(7):753-60.
7. Erman, A, et al. 2009. Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development. J Histochem Cytochem. 57: 721-730.
8. Escribano, C, et al. 2009. CCR7-Dependent Stimulation of Survival in Dendritic Cells Involves Inhibition of GSK3β. J Immunol. 183: 6282-6295.
9. 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).
10. 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). 
11. 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).
12. 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).
13. Altmeyer, A., et al. Cell death after high-LET irradiation in orthotopic human hepatocellular carcinoma in vivo. In Vivo. 25: 1-9 (2011).
14. 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).
15. 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).
16. 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).
17. Delgado-Martín, C., Escribano, C., Pablos, J.L., Riol-Blanco, L., and 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).

Irradiation-induced Apoptosis Detected in vivo with SR-FLIVO™

After administering 0Gy and 15Gy whole lung doses of gamma radiation, Eric Hernady at the University of Rochester Medical Center in Rochester, NY labeled activated caspases in mouse lung parenchymal cells in vivo with SR-FLIVO (cat. #983).
 
Six hours post-irradiation, 100µl of SR-FLIVO was injected via the tail vein into experimental (a) and control (b) mice. The SR-FLIVO reagent circulated for 18 hours prior to sacrifice. Mice received an intracardiac perfusion with heparinized saline followed by zinc-buffered formalin. Tissues were processed, paraffin-embedded, sectioned (5µm), counterstained with DAPI, and imaged with a fluorescent microscope (40X).

Active caspases appear as a bright red cytoplasmic stain with discrete blue nuclei.

In vivo assessment of experimental chemotherapy: apoptotic tumor cells fluoresce red

These pictures were taken of live tumors growing inside 2 different mice after injection with SR-FLIVO (cat. #983). The control mouse (left) received a placebo while the test mouse (right) was treated with ATO. 24 hours after treatment, both mice were injected intravenously with ICT’s red SR-FLIVO probe in the tail vein. Pictures were taken 30 minutes later. The control tumor exhibits some level of apoptosis as expected, whereas the ATO-treated mouse tumor exhibits a much higher level of apoptosis. Areas with high levels of active caspases, and thus more bound fluorescence, appear as overexposed white spots in the image. Black areas are living tumor cells. To quantify the level of caspase activity and apoptosis, Dr. Griffin excised the tumor, trypsinized the cells, and analyzed them on a flow cytometer (below).

Excised tumors analyzed via flow cytometry 

Cell suspensions were made from excised mammary tumors (like those above) and analyzed on a flow cytometer. After labeling both tumors with FLIVO, the level of apoptosis could be quantified: it doubled in SCK mammary tumors after treatment with ATO. 39% of tumor cells treated with ATO were apoptotic; 18% of untreated tumor cells were apoptotic. Data courtesy of Dr. Robert Griffin, Ph.D. University of Minnesota Medical School, Department of Radiation Oncology (now at the University of Arkansas); flow cytometry performed by Dr. Michael Olin, Ph.D, University of Minnesota.

Non-invasively image apoptosis

FLIVO™ allows you to non-invasively image apoptosis in whole live animals. Human colon carcinoma COLO205 cells were injected S/C into female nude mice (left, tumor circled). After 27 days, animals were treated with a control or TRAIL at 25 mg/Kg (right), then injected with SR-FLIVO™. TRAIL induces apoptosis within 1 hour, and the level of apoptosis significantly increased by 4-hours (right) and 24-hours post-treatment (data not shown). COLO205 tumor cells are being killed - they fluoresce brightly with SR-FLIVO™. Data courtesy of Dr. Peter Lassota, Caliper Life Sciences / Xenogen (preliminary data without systems optimization shown). Please call ICT for updates on this technology - 800-829-3194.

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. 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.

How many tests can be run with the trial size and regular size kits?
The necessary amount of FLIVO will vary by experiment. The trial size FLIVO kit provides enough reagent for 4-10 injections, 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. The regular size FLIVO kit provides 4x as much reagent as the trial size kit.

Can FLIVO be imaged non-invasively?
Preliminary data suggest that it is possible to image SR-FLIVO™ (catalog #983) with a non-invasive optical imaging system. Positive results have been achieved using SR-FLIVO™ with the Caliper/Xenogen IVIS machine. The red SR fluorophore will penetrate up to 3 mm of skin tissue for detection with IVIS; this works best on hairless animals with light skin. A final protocol has not been set yet regarding this technique; please contact ICT for updates.

ICT is developing additional tracers that will be more highly compatible with non-invasive optical imaging systems as well as probes for alternative detection methods.

Can I read SR-FLIVO™ labeled cells using a flow cytometer?
We recommend that the investigator use a flow cytometer that includes a green or yellow laser excitation option for flow cytometry analysis of SR-FLIVO stained cells. SR-FLIVO labeled cells may be detected using the conventional blue laser (488 nm) excitation setting common to most flow cytometers, however, the efficiency of sulforhodamine B excitation at this wavelength is very low, leading to a dramatic reduction in assay sensitivity.

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