FAM-FLIVO® in vivo Apoptosis Kits

Catalog Number: 980 (small); 981 (large)

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Sale prices on FAM-FLIVO are automatically applied to online orders. To receive this special pricing offline (fax, email, phone orders), simply reference FLIVO20 as part of our 20 Years of Solutions Anniversary Promotion!

Notice: Pursuant to the sale of FLIVO technology (see Press Release), FLIVO® products will be discontinued from the ImmunoChemistry Technologies catalog 1/1/15. Products will be available for sale until December 31, 2014, while supplies last. The products will be unavailable from ICT after 12/31/14.

FLIVO® in vivo Apoptosis Kits provide a simple yet accurate method of in vivo apoptosis detection. The non-cytotoxic FLIVO (FLuorescence in vIVO) probes are injectable fluorescent inhibitors of caspase activity. Upon intravenous injection, the FLIVO® probes preferentially form covalent bonds with active caspases, marking cells undergoing apoptosis with fluorescence. The bound FLIVO® probe will remain inside the cell as long as the cell membrane is intact, marking the cell for apoptosis detection. Any unbound FLIVO® in vivo apoptosis detection probe is removed with the natural circulation of the animal. The resulting fluorescent signal within samples is a direct measure of apoptosis that occurred at the time the reagent was injected.

FAM-FLIVO® has a green fluorescent label, carboxyfluorescein, with an optimal excitation range from 490 - 495 nm and optimal emission range from 515 - 525 nm. FLIVO® is available in four fluorescent analogs: FAM-FLIVO (green fluorescence, carboxyfluorescein); SR-FLIVO (orange-red fluorescence, sulforhodamine B); NIR-FLIVO 690 (near-infrared fluorescence); and NIR-FLIVO 747 (near-infrared fluorescence). Visible fluorescence FLIVO assays are supplied in small kits (enough for 6 mice or rats) and large kits (enough for 24 mice or rats). Near-infrared fluorescence NIR-FLIVO assays are supplied as vials (10 tests each) or kits (20 tests).

Green FAM FLIVO® in vivo Apoptosis Kit

As part of our 20 Years of Solutions Anniversary Promotions, we are offering a 20% discount on all FLIVO products through 11/30/14. To receive this special pricing online or offline (fax, email, phone orders), simply enter or reference FLIVO20 with your order.

Notice: Pursuant to the sale of FLIVO technology IP (see Press Release), FLIVO® products will be discontinued from the ImmunoChemistry Technologies catalog 1/1/15. Products will be available for sale until December 29, 2014, while supplies last.


FLIVO® in vivo Apoptosis Kits

FLIVO assays provide a simple yet accurate method of in vivo apoptosis detection. 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. ICT's FLIVO® tracers 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 on other tracers.

Simple and 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 cells undergoing apoptosis with fluorescence. The bound FLIVO® probe will remain inside the cell as long as the cell membrane is intact, marking these targets for apoptosis detection. Any unbound FLIVO is removed from the circulation of the animal in about an hour, clearing the body through the kidneys and liver. The remaining green fluorescent signal in the tissue after clearance is a direct measure of apoptosis via caspase activity that occurred at the time the reagent was injected.

Eliminate False Positives

Once unbound FLIVO® in vivo apoptosis detection reagent has been allowed to clear from the non-apoptotic tissues, the tissues are ready for analysis and no further staining is necessary. Because FLIVO® is a direct in vivo apoptosis detection probe, it eliminates false positives that may arise from ex vivo manipulation and staining. This gives a true representation of the induction of in vivo apoptosis 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.


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 (see Citations and Data tabs). 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.

Future Clinical Applications

After years of research, the FLIVO® technology has been sold for further development in clinical applications. Diagnostic detection methods may be developed to more accurately assess tumor shrinkage, neurodegeneration, retinal degeneration, and other disease states in both research and clinical settings. Our research customers continue to find 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 inform doctors and their patients if their condition is improving.

Related Apoptosis Tracers

FLIVO® is available in four fluorescent analogs: FAM-FLIVO (green fluorescence, carboxyfluorescein); SR-FLIVO (orange-red fluorescence, sulforhodamine B); NIR-FLIVO 690 (near-infrared fluorescence); and NIR-FLIVO 747 (near-infrared fluorescence). Visible fluorescence FLIVO assays are supplied in small kits (enough for 6 mice or rats) and large kits (enough for 24 mice or rats). Near-infrared fluorescence NIR-FLIVO assays are supplied as vials (10 tests each) or kits (20 tests).

Product Manuals:

FAM FLIVO in vivo Apoptosis kit manual.pdf

Reagent Name: FAM-FLIVO™

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.



  1. 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).
    Arsenic trioxide (ATO, Trisenox) is a potent anti-vascular agent and significantly enhances hyperthermia and radiation response. To understand the mechanism of the anti-tumor effect in vivo we imaged the binding of a fluorescently-labeled poly-caspase inhibitor (FLIVO) in real time before and 3 h or 24 h after injection of 8 mg/kg ATO. FSaII tumors were grown in dorsal skin-fold window chambers or on the rear limb and we observed substantial poly-caspase binding associated with vascular damage induced by ATO treatment at 3 and 24 h after ATO injection. Flow cytometric analysis of cells dissociated from the imaged tumor confirmed cellular uptake and binding of the FLIVO probe. Apoptosis appears to be a major mode of cell death induced by ATO in the tumor and the use of fluorescently tagged caspase inhibitors to assess cell death in live animals appears feasible to monitor and/or confirm anti-tumor effects of therapy. PMID: 17994796
  2. Cursio, R, et al. 2008. Liver Apoptosis Following Normothermic Ischemia-Reperfusion: In VivoEvaluation of Caspase Activity by FLIVO Assay in Rats. Transplant P. 40, 2038-2041.
    Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular apoptosis. Caspase activation is involved in the initiation and execution of apoptosis. The aim of this study was to determine in vivo caspase activity in normothermic liver I-R in rats. Segmental normothermic ischemia of the liver was induced for 120 minutes in rats. After intravenous injection of the green probe FLIVO, in vivo caspase-3- and -7-specific activity was determined using fluorescence microscopy, in either nonischemic or ischemic liver lobes at 3 and 6 hours after reperfusion. Liver apoptosis was assessed by the deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Fluorescence microscopy showed that in vivo caspase-3- and -7-specific activities were significantly increased (P< .005) in ischemic lobes at 3 and 6 hours of reperfusion, compared with nonischemic liver lobes. Quantitative analysis of apoptotic cells measured by the TUNEL method showed a significant increase among apoptotic cells in ischemic lobes at 3 and 6 hours after reperfusion (P< .005), compared with nonischemic liver lobes. In conclusion, 120-minute normothermic liver I-R resulted in increased caspase-3- and -7-specific activities and in liver cell apoptosis. PMID: 18675124
  3. 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.
    Apoptosis detection methodology is an ever evolving science. The caspase family of cysteine proteases plays a central role in this environmentally conserved mechanism of regulated cell death. New methods that allow for the improved detection and monitoring of the apoptosis-associated proteases are key for further advancement of our understanding of apoptosis-mediated disease states such as cancer and Alzheimer's disease. From the use of membrane permeant fluorescent-labeled inhibitors of caspases (FLICA) probe technology, we have demonstrated their successful use as tools in the detection of apoptosis activity within the in vitro and in vivo research setting. In this chapter, we provide detailed methods for performing in vitro apoptosis detection assays in whole living cells, using flow cytometry, and 96-well fluorescence plate reader analysis methods. Furthermore, novel flow cytometry-based cytotoxicity assay methods, which incorporate the FLICA probe for early apoptosis detection, are described. Inclusion of this sensitive apoptosis detection probe component into the flow-based cytotoxicity assay format results in an extremely sensitive cytotoxicity detection mechanism. Lastly, in this chapter, we describe the use of the FLICA probe for the in vivo detection of tumor cell apoptosis in mice and rats. These early stage in vivo-type assays show great potential for whole animal apoptosis detection research. PMID: 18175816
  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.
    BACKGROUND:  Phosphoregulation of signal transduction pathways is a complex series of reactions that may modulate the cellular response to ischemia-reperfusion (I-R). The aim of this study was to evaluate the effect of normothermic liver I/R-induced apoptosis on phosphorylation and activation of signal proteins in tyrosine kinase pathways.
    MATERIALS AND METHODS: In rats, a segmental normothermic ischemia of the liver was induced for 120 min. Liver apoptosis was determined using terminal deoxynucleotide-transferase-mediated deoxyuridine triphosphate nick end labeling assay, and activity of caspases-3 and -7 was determined by fluorescence. Liver tyrosine phosphorylation of proteins was examined by Western blot analysis.
    RESULTS:  Normothermic I-R resulted in increased in vivo caspases-3 and -7 activity and in liver apoptosis. Shc tyrosine phosphorylation and activation of ERK1/2 were increased after reperfusion, while tyrosine phosphorylation of IRS-1 and activation of PKB/Akt were decreased.
    CONCLUSIONS:  Normothermic liver I-R leads to increased apoptosis and to modifications in protein tyrosine phosphorylation pathways.
    PMID: 18679708
  5. 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.
    This protocol describes a method to detect and measure the percentage of apoptotic or live dendritic cells (DCs) in popliteal lymph nodes (PLNs). DCs labelled with fluorescent cell trackers are subcutaneously (s.c.) injected in mice. After allowing the DCs to reach the PLNs, animals are intravenously injected with FLIVOTM, a permeant fluorescent reagent that selectively marks apoptotic cells. Of note, the fluorophore moiety of the latter reagent should be selected so as its emission wavelength can be distinguished under the confocal microscope from that of the fluorescent cell tracker used to label the cells. Explanted PLN are then examined under a two-photon microscope to analyse for the presence of apoptotic cells among the DCs injected. 
    Caspases are the key proteolytic components of the demolition machinery that cleaves vital cell substrates during apoptosis1. These proteases display in their active center a cysteine residue that is required for their activity1. Active caspases can be detected in situ by Fluorochrome-Labeled Inhibitors of CAspases (FLICA)2-5, a technique based on the fact that fluorochrome-labeled inhibitors of caspases, through its conjugated reactive fluoromethyl-ketone (fmk) moiety, can form an irreversible thio-methyl ketone link with the cystein in the active center of these enzymes. Importantly, reagents based on FLICA do not bind to pro-caspases or any inactive form of these enzymes. Specifically, to detect apoptotic DCs in vivo we have used FLIVOTM, a reagent based on FLICA that is composed of a poly caspase binding inhibitor probe (Val-Ala-Asp(OMe)-fluoromethyl ketone (VAD-FMK)) which binds irreversibly to apoptotic6 caspases-1, -3, -4, -5, -6, -7, -8 and -9, conjugated to fluorescent dyes (either red SR-FLIVO or green FAM-FLIVO)7. These reagents can be intravenously injected to live mice and, since they are cell permeant, they can be used to stain apoptotic DCs in the LNs. In non-apoptotic DCs, which lack caspases, FLIVO reagents are not retained and leak out. However, in apoptotic DCs they form covalent bonds with intracellular caspases, resulting in the trapping of the (green or red) FLIVOTMfluorescent signal within these cells. Subsequently, the LNs can be explanted and examined by two photon microscopy to search for the presence of fluorescent FLIVO, indicating apoptosis, in the DCs.
  6. Erman, A, et al. 2009. Apoptosis and desquamation of urothelial cells in tissue remodeling during rat postnatal development. J Histochem Cytochem. 57: 721-730.
    Postnatal rat urothelium was studied from day 0 to day 14, when intense cell loss as part of tissue remodeling was expected. The morphological and biochemical characteristics of urothelial cells in the tissue and released cells were investigated by light and electron microscopy, by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, by annexin V/propidium iodide assay, and by immunofluorescent detection of active caspases and tight-junction protein occludin. Intense apoptosis and massive desquamation were detected between postnatal days 7 and 10. During this period, active caspases and TUNEL-positive cells were found in the urothelium. Disassembled cell-cell junctions were detected between cells. The majority of desquamated cells expressed no apoptotic cell morphology, but were active caspase positive and TUNEL positive. Ann+/PI- apoptotic bodies and desquamated Ann+/PI+ cells were detected in the lumen. These results indicate that apoptosis and desquamation participate in urothelial cell loss in the rat early postnatal period, indispensable for fast urothelial remodeling during development. PMID: 19365092
  7. Olin, M., Roy, S., and Molitor, T. 2010. In Vivo Morphine Treatment Synergistically Increases LPS-Induced Caspase Activity in Immune Organs. J Neuroimmune Pharmacol. 5, 4:546-552.
    Caspases are a family of proteins important for the elimination of infected cells through the induction of apoptosis as well as the initiation of inflammatory cytokines including IL-1β and IL-18. Morphine exposure to animals and/or cells has been associated with the induction of apoptosis. The most common practices of apoptosis detection have involved removing tissues from animal or humans and the analysis of apoptosis on cells or tissues. These methods can potentially induce spontaneous apoptosis that is unrelated to the actual treatment. The objective of this study was to develop an in vivo detection method for assessing caspase activity induced both by morphine directly and by morphine combined with lipopolysaccharide (LPS)-immune activation. Mice were administered saline, morphine, LPS, or a combination of morphine and LPS. Prior to sacrifice, mice were injected with a polycaspase-specific apoptosis detection probe to detect internal caspase activity in vivo. Results revealed that morphine alone did not directly induce caspase activity. However, morphine significantly enhanced the LPS-induced caspase activity in spleen, thymus, and bone marrow-derived immune cells. The use of a poly-caspase detection probe methodology to label caspase activity in vivo provides a powerful quantitative tool for the in vivo analysis of caspase activity. PMID: 20390371
  8. Cursio, R, et al. 2010. Induction of Different Types of Cell Death After Normothermic Liver Ischemia-Reperfusion. Transplant P. 42, 3977-3980.
    Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular autophagy, apoptosis, and necrosis. The aim of this study was to investigate these three types of cell death in normothermic liver I-R in rats. A segmental normothermic ischemia of the liver was induced for 120 minutes. Liver autophagy was evaluated by transmission electron microscopy and LC3 (Light Chain 3) immunohistochemical studies. Liver apoptosis was assessed by FLIVO (FLuorescence in vIVO) and TUNEL (TdT-mediated dUTP nick end labeling) assays. Liver necrosis was determined by optical microscopic examination. Autophagy was increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Fluorescence microscopy showed in situ caspase-3 and -7 specific activity to be increased in ischemic liver lobes after 6 hours of reperfusion, compared with nonischemic lobes. Quantitative analysis of apoptotic cells evaluated by the TUNEL method showed a clearly significant increase in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Necrotic cell death was significantly increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes (P < .005). In conclusion, 120 minutes normothermic liver I-R resulted in increased autophagic, apoptotic and necrotic cell death. PMID: 21168604
  9. Altmeyer, A, et al. 2011. Cell Death After High-LET Irradiation in Orthotopic Human Hepatocellular Carcinoma in vivo. In Vivo, 25: 1 - 9.
    Hepatocellular carcinoma (HCC) represents the sixth most common cancer worldwide and a major health problem since the choice of treatment is limited due to chemo- and radio-resistance. It was previously reported that high linear energy transfer (LET) radiation induced massive autophagic cell death in the human HCC SK-Hep1 cell line in vitro. This study analyzed the effects of high-LET radiation on the same HCC tumor model, orthotopically transplanted into nude mice. For this purpose, after surgical xenograft in the liver, animals were irradiated with fast neutrons and cell death occurring in the tumors was assessed with various techniques, including electron microscopy and probe-based confocal laser endomicroscopy. Results indicate that considerable autophagy and only limited apoptosis took place in the tumor xenografts after high-LET irradiation. These data confirm the previous in vitro results, suggesting that autophagy may act as a predominant mode of cell death in the efficacy of high-LET radiation. PMID: 21282728
  10. Darzynkiewicz, Z., Pozarowski, P., Lee, B.W., and Johnson, G.L. 2011. 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. 
    Activation of caspases is a hallmark of apoptosis. Several methods, therefore, were developed to identify and count the frequency of apoptotic cells based on the detection of caspases activation. The method described in this chapter is based on the use of fluorochrome-labeled inhibitors of caspases (FLICA) applicable to fluorescence microscopy, and flow- and image-cytometry. Cell-permeant FLICA reagents tagged with carboxyfluorescein or sulforhodamine when applied to live cells in vitro or in vivo, exclusively label cells that are undergoing apoptosis. The FLICA labeling methodology is simple, rapid, robust, and can be combined with other markers of cell death for multiplexed analysis. Examples are presented on FLICA use in combination with a vital stain (propidium iodide), detection of the loss of mitochondrial electrochemical potential, and exposure of phosphatidylserine on the outer surface of plasma cell membrane using Annexin V fluorochrome conjugates. Following cell fixation and stoichiometric staining of cellular DNA, FLICA binding can be correlated with DNA ploidy, cell cycle phase, DNA fragmentation, and other apoptotic events whose detection requires cell permeabilization. The "time window" for the detection of apoptosis with FLICA is wider compared to that with the Annexin V binding, making FLICA a preferable marker for the detection of early phase apoptosis and more accurate for quantification of apoptotic cells. PMID: 21057924
  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). 
    S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization. PMID: 21785166
  12. Greenblatt, S, et al. 2012. Knock-in of a FLT3/ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model. Blood. 119:2883-2894. 
    Constitutive activation of FLT3 by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML). FLT3/ITD mutations have also been observed in myelodysplastic syndrome patients both before and during progression to AML. Previous work has shown that insertion of an FLT3/ITD mutation into the murine Flt3 gene induces a myeloproliferative neoplasm, but not progression to acute leukemia, suggesting that additional cooperating events are required. We therefore combined the FLT3/ITD mutation with a model of myelodysplastic syndrome involving transgenic expression of the Nup98-HoxD13 (NHD13) fusion gene. Mice expressing both the FLT3/ITD and NHD13 transgene developed AML with 100% penetrance and short latency. These leukemias were driven by mutant FLT3 expression and were susceptible to treatment with FLT3 tyrosine kinase inhibitors. We also observed a spontaneous loss of the wild-type Flt3 allele in these AMLs, further modeling the loss of the heterozygosity phenomenon that is seen in human AML with FLT3-activating mutations. Because resistance to FLT3 inhibitors remains an important clinical issue, this model may help identify new molecular targets in collaborative signaling pathways. PMID: 22323452
  13. Griffin, RJ, et al. 2012. Vascular Disrupting Agent Arsenic Trioxide Enhances Thermoradiotherapy of Solid Tumors. J Oncol, vol. 2012, Article ID 934918, 7 pages, doi:10.1155/2012/934918 
    Our previous studies demonstrated arsenic trioxide- (ATO-) induced selective tumor vascular disruption and augmentation of thermal or radiotherapy effect against solid tumors. These results suggested that a trimodality approach of radiation, ATO, and local hyperthermia may have potent therapeutic efficacy against solid tumors. Here, we report the antitumor effect of hypofractionated radiation followed by ATO administration and local 42.5 °C hyperthermia and the effects of cisplatin and thermoradiotherapy. We found that the therapeutic efficacy of ATO-based thermoradiotherapy was equal or greater than that of cisplatin-based thermoradiotherapy, and marked evidence of in vivo apoptosis and tumor necrosis were observed in ATO-treated tumors. We conclude that ATO-based thermoradiotherapy is a powerful means to control tumor growth by using vascular disruption to augment the effects of thermal and radiation therapy. PMID: 22272199
  14. Goetz, M, Ansems, JV, Galle, PR, Schuchmann, M, and Kiesslich, R. In vivo real-time imaging of the liver with confocal endomicroscopy permits visualization of the temporospatial patterns of hepatocyte apoptosisAm J Physiol Gastrointest Liver Physiol. 2011; 301:G764-G772. [Abstract]
  15. Meng, F and Alpini, G. Confocal laser endomicroscopy in dynamic evaluation of hepatic apoptosis in vivo. Am J Physiol Gastrointest Liver Physiol. 2011; 301:G762-G763. [Full Text]


See our SR-FLIVO® in vivo apoptosis kit page for additional FLIVO Citations.

Apoptosis in Tumors Labeled in vivo Before and After Treatment with ATO.

Cell death in tumors detected in vivo before and after chemotherapy.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.

Apoptosis detected in vivo in rat liver with FLIVO #981.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 Labeled In Vivo For Caspase Activity in Diabetic Rat Brain.

Dying neurons are revealed using FAMFLIVO in vivo 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.

Apoptosis in chick brain revealed by in vivo staining with FAM FLIVO.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

Apoptosis in bone marrow after morphine LPS treatment using FLIVO.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.










Target: apoptosis, in vivo apoptosis
Excitation / Emission: 488 nm / 530 nm
Method of Analysis: Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader
Types of Samples: animal studies
Kit Contents:

Kit #980:

  • FAM FLIVO in vivo apoptosis probe, 1 vial x 52 ug
  • 10x Injection Buffer, 5 mL
  • Manual

Kit #981:

  • FAM FLIVO in vivo apoptosis probe, 4 vials x 52 ug each
  • 10x Injection Buffer, 5 mL
  • Manual
Storage: 2°-8° C


Certificates of Analysis: Call 1-800-829-3194 or 1-952-888-8788

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.

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