Green FAM FLIVO™ In Vivo Apoptosis Kit

FLIVO™ (FLuorescence in vIVO) probes 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™ probes preferentially form covalent bonds with active caspases, causing cells undergoing apoptosis to fluoresce.  FAM-FLIVO™ (FAM-VAD-FMK), a green fluorescent probe, is a carboxyfluorescein (FAM)-conjugated valylalanylaspartic acid (VAD) fluoromethyl ketone (FMK).  FLIVO™ is also available with a visible red fluorescent label (SR) 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 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 (see manual, Figure 1). We advise against paraffin-embedding samples labeled with green FAM-FLIVO. Tissues labeled with FAM-FLIVO™ may be counter-stained with cellular imaging reagents, such as red Nissl (manual, Figure 2), blue DAPI (manual, Figure 3), or DRAQ5, and fixed or frozen for future analysis. The fluorescence intensity can be quantified by excising the tissue and analyzing cells with a flow cytometer (manual, Figure 4). FAM-FLIVO™ excites at 488 nm and emits at 530 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 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 FAM-FLIVO, select your desired size from the drop-down menu above, and click on the green "Add to Cart" button.

Catalog no. 980: FAM-FLIVO™ in vivo Apoptosis Kit, green fluorescence, small; $179 USD
Catalog no. 981: FAM-FLIVO™ in vivo Apoptosis Kit, green 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 probe, 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.

FAM-FLIVO™ CITATIONS

1. Tsai, YC, et al. 2007. The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nat Med. 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. Lee, B.W., Olin, M.R., Johnson, G.L., and Griffin, R.J. 2008. In vitro and in vivo apoptosis detection using membrane permeant fluorescent-labeled inhibitors of caspases. Methods Mol Biol. 414:109-35.
5. 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.
6. Delgado-Marti­n, 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
7. 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.
8. Erman, A, et al. 2009. Apoptosis and desquamation of urothelial cells in tissue remodeling during rat postnatal development. J Histochem Cytochem. 57: 721-730.
9. Escribano, C, et al. 2009. CCR7-Dependent Stimulation of Survival in Dendritic Cells Involves Inhibition of GSK3β. J Immunol. 183: 6282-6295. [Full Text] 
10. Altmeyer, A, et al. 2011. Cell Death After High-LET Irradiation in Orthotopic Human Hepatocellular Carcinoma in vivo. In Vivo, 25: 1 - 9.
11. 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]

Newst Citations describe use of FAM-FLIVO 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]

See our SR-FLIVO page for additional FLIVO Citations: http://www.immunochemistry.com/products/detection-reagents/flivo/red-sr-flivo-in-vivo-apoptosis-kit.html

Tumor cell death labeled in vivo before and after treatment with ATO.

These images were taken through a window chamber of one live FSaII murine fibrosarcoma tumor (in vivo). Before any treatment was administered, FAM-FLIVO™ (catalog #981) was injected into the mouse to determine how many of the tumor cells were apoptotic: caspase-positive cells turn green, caspase-negative cells are dark. The first picture (left) was taken 45 minutes after FAM-FLIVO™ was injected. Very few of the cells turned green, indicating that most of the tumor is living. The mouse was then injected with a drug (arsenic trioxide, ATO) for 3 hours. FAM-FLIVO™ was again injected into the mouse, and the second picture (right) was taken 45 minutes later. Within a few hours of treatment, Dr. Robert Griffin (formerly at the U of MN, now at the U of AR) was able to assess the efficacy of the drug using FAM-FLIVO™: this dose of ATO induced apoptosis in most of the tumor cells.

Apoptosis revealed in rat liver ischemia using FAM-FLIVO.

In this example, apoptotic ischemic rat liver cells fluoresce bright green after in vivo labeling with FAM-FLIVO™ compared to non-ischemic hepatic tissue. In the experimental rat (left), segmented normothermic ischemia of the liver was induced for 120 minutes. Six hours post reperfusion, FAM-FLIVO™ (catalog #981) was injected into the portal vein and allowed to circulate for 10 minutes prior to sacrifice. FLIVO™ was also injected into the portal vein of the healthy control (right). 5 µm cryosections of liver were prepared, and nuclei were visualized in blue using DAPI. A brighter green signal clearly stands out from the hepatocytes containing active caspases that are distributed around the vessel in the ischemic condition compared to the control (non-ischemic) tissue. Data courtesy of Drs. Raffaele Cursio and Pascal Colosetti, INSERM U895 C3M E2, Nice, France.

Dying neurons in diabetic rat brain.

These images reveal healthy and apoptotic neurons of the periaqueductal gray (PEG) of control S/D rats (healthy, left) and 8-week STZ diabetic S/D rats (apoptotic, right). 30 minutes prior to sacrifice, FAM-FLIVO™ (catalog #981) was injected IV. 20 micron frozen sections were prepared and stained with red Nissl to visualize all neurons. Using FAM-FLIVO™ to quantitate caspase activity in vivo, it is shown that diabetes causes a significant increase in apoptosis in the neurons of the PEG. Data courtesy of Dr. Thomas Morrow, University of Michigan, Ann Arbor.

Dying neurons in chick brain.

As part of his thesis work to examine naturally-occurring neuron death in seasonally-manipulated songbirds, Mr. Chris Thompson at the University of Washington, Seattle used FAM-FLIVO™ (catalog # 981) to assess apoptosis in vivo. Mr. Thompson injected staurosporine (catalog #6212, a protein kinase inhibitor that induces apoptosis) into the forebrain of a female house sparrow. ~20 hours later, he injected FAM- FLIVO™ intravenously into the jugular. 30 minutes later, he sacrificed the bird via transcardial perfusion with heparinized saline and 4% paraformaldehyde. He postfixed the brain for 48 hours, embedded it in gelatin, postfixed and cryoprotected the brain in 10% NBF and 20% sucrose for 48 hours more. 40 um slices of the brain were made on a freezing microtome, and sections were mounted onto slides and coverslipped with ProLong antifade mountant. Neurons with active caspases fluoresce green. This picture shows one apoptotic neuron at 100X.

Apoptosis in bone marrow after morphine treatment

C57BL/S126 mice were treated with morphine and/or LPS, or a placebo for 48 hours. FAM-FLIVO was injected in the tail vein 45 minutes prior to sacrifice. Following sacrifice, bone marrow cells were obtained and analyzed by flow cytometry. The data demonstrate an increase in apoptosis induction in the bone marrow leukocytes of morphine treated animals. Data courtesy of Dr. Mike Olin, University of Minnesota.

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?
FAM-FLIVO is not optimal for noninvasive imaging of apoptotic tissues.  Our new near-infrared (NIR) fluorescent apoptosis tracers, NIR-FLIVO, may be imaged non-invasively in live animals with small animal optical imaging systems.
ICT is developing additional tracers that will be compatible with alternative non-invasive detection methods.

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