FLIVO® kits provide a simple and accurate method to detect caspase activity in vivo. Similar to our FLICA® probes but optimized for whole live animal labeling. To label cells containing active caspases, inject FLIVO intravenously and let it circulate ~60 minutes. Because the reagent is cell-permeant, it readily diffuses in and out of all cells that it encounters as it circulates throughout the body. If there are active caspase enzymes inside a cell, FLIVO will form an irreversible covalent bond with the caspase active site. The bound FLIVO probe will remain inside the cell if 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. Once the excess FLIVO has cleared from the body of the animal, the tissues are ready for analysis. No further staining is necessary. Tissues can be viewed directly through a window chamber system or other accessible cavity. Alternatively, target tissues can be removed and processed for analysis. FLIVO is very sensitive and will pick up naturally occurring background apoptosis. Apoptotic cells have more active caspases than control cells, therefore they fluoresce brighter with FLIVO.
FAM-FLIVO® In vivo Poly Caspase Assay
$184.00 – $504.00
FLIVO® (FLuorescence in vIVO) is a powerful method for assessing caspase activity in vivo. FAM-FLIVO poly caspase probes are non-cytotoxic, cell-permeant fluorescent inhibitors of caspases optimized for use in whole live animals. ICT’s FAM-FLIVO® poly caspase inhibitor probe contains the preferred binding sequence for most caspases (Val-Ala-Asp or VAD). This preferred poly caspase tripeptide binding sequence is labeled with a carboxyfluorescein (FAM) dye and a fluoromethyl ketone (FMK) reactive entity.
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1. Prepare samples and controls.
2. Dilute 10X Injection Buffer 1:10 by adding 45 mL diH20.
3. Reconstitute FAM-FLIVO with 50 µL DMSO.
4. Dilute FAM-FLIVO 1:12 with 550 µL 1X Injection Buffer.
5. Inject 100 µL intravenously.
6. Let FAM-FLIVO circulate 30-60 minutes.
7. View live tumor through a window chamber using a fluorescence microscope.
8. If not viewing directly, excise tissue.
9. If desired, label with additional stains, such as Hoechst 33342, or an antibody.
10. If desired, fix cells.
11. Analyze with a fluorescence microscope, flow cytometer, or a window chamber system. FAM-FLIVO excites at 492 nm and emits at 520 nm.
• Reagent Name: FAM-FLIVO® Poly Caspase Inhibitor (FAM-VAD-FMK)
• Target: Poly Caspase
• Excitation/Emission: 492 nm/520 nm
• Method of Analysis: Fluorescence Microscope, Flow Cytometer, Window Chamber System
• Types of Samples: Whole live animal, excised tissue
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping
CASPASE ACTIVITY IN RAT BRAIN
Using FAM-FLIVO to monitor cell death, there is a clear distinction between healthy and apoptotic neurons. In this live animal brain study of diabetes, Dr. Thomas Morrow at the University of Michigan VAMC Ann Arbor was able to assess neurodegeneration via caspase activity in control (left) and 8-week STZ diabetic rats (right). 30 minutes prior to sacrifice, FAM-FLIVO (catalog #981) was injected intravenously to directly label caspase-positive apoptotic neurons. After sacrifice, 20 µm frozen sections of the periaqueductal gray (PAG) were prepared and counter-stained with red fluorescent Nissl to identify all neurons. Dying apoptotic neurons exhibit dual staining with FAM-FLIVO (yellow/green) and Nissl (red). In this model, diabetic animals show greater levels of caspase activity in the PAG than control animals.
ISCHEMIA IN LIVER
Using FLIVO to analyze ischemia in liver, Drs. Raffaele Cursio and Pascal Colosetti (INSERM U895 C3M E2, Nice, France) were able to identify hepatocytes in which caspases were activated. Segmental normothermic ischemia of the liver was induced for 120 minutes. Six hours post-reperfusion, FAM-FLIVO (catalog #981) was injected into the portal veins of the rats and left to circulate for 10 minutes. Images were taken from 5 μm cryosections of liver with the nuclei visualized in blue using DAPI (400X). A bright green signal is clearly seen in hepatocytes containing active caspases distributed around the vessel in the ischemic condition (right) compared to the control non-ischemic tissue (left).
IN VIVO DETECTION OF APOPTOSIS OF ACTIVE CASPASES IN UROTHELIAL CELLS.
The reagent was diluted in dimethyl sulfoxide and sterile injection buffer, and 40 ml of prepared reagent solution was injected intravenously through the femoral vein of a young rat. After circulation for 30 min, the urinary bladder was excised. Some fully filled and stretched urinary bladders were fixed in 2% paraformaldehyde and cut into flat pieces, and the others were immediately frozen and cut into 7-mm-thick frozen sections. Flat tissue pieces and frozen sections were mounted in the mounting medium Vectashield with DAPI (Vector Laboratories) and observed with a Nikon Eclipse TE 300 fluorescence microscope. An area of intact three-layered urothelium shows caspase activity (green). Tissue section on postnatal day 10. Basal lamina is marked with a dotted line. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). L, lumen of the urinary bladder. Bar = 10 mm. Reference: Volume 57(8): 721–730, 2009 Journal of Histochemistry & Cytochemistry Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development. Andreja Erman, Daša Zupančič, and Kristijan Jezernik. Institute of Cell Biology, Faculty of Medicine, Ljubljana, Slovenia
OPTICAL NERVE TRANSECTION RAT RETINA
FAM-FLIVO was injected intravitreally after Optical Nerve Transection ONT procedure at 20X. Fluorescent spots indicate apoptotic RGC’s Retinal Glial/Ganglia Cells are seen in the experimental eye (left), which are absent in the contralateral eye (right). Retina with macular degeneration vs. healthy. Data courtesy of BL. A. Labs 2006
BRAIN DAMAGE IN DIABETIC RATS
On the left is the control rat, on the right is the STZ diabetic (8-weeks old) rat. 30 minutes before sacrificing the animals, FAM-FLIVO was injected IV, then 20um frozen sections of the periaqueductal grey (PAG) were prepared and counter-stained with Nissl, which is red fluorescent, to identify all neurons. Dying apoptotic neurons in the brain section appears yellow/green with FAM FLIVO. In this model, diabetic animals show greater levels of caspase activity in the PAG than control animals. Unpublished data courtesy of Dr. Thomas Morrow at the University of Michigan VAMC Ann Arbor.
IN VIVO APOPTOSIS IN CHICK BRAIN
FLIVO was injected 35 minutes prior to sacrifice. In the control bird (left), you can see a low level of uniform background staining. In the experimental chick (right), nearly every auditory neuron in the deafferented NM is brightly stained with FAM-FLIVO. Caspases are activated in every neuron of the NM just 6 hours after the cochlea is removed. If the chick can’t hear, the brain dies. Unpublished data courtesy of Ms. Yuan Wang, University of Washington.
IN VIVO APOPTOSIS DETECTION
Arsenic trioxide (ATO)-induced apoptosis in FSaII murine fibrosarcoma tumors was detected in vivo using FAM-FLIVO. Skin-fold window chambers were surgically implanted into dorsal skin folds on female nu/nu mice. FSaII fibrosarcoma cells were added to skin then glass windows were placed to allow viewing. When tumor growth was apparent, mice were injected with PBS (negative control, left) or 8 mg/kg ATO (apoptosis inducing agent, right). FAM-FLIVO (~10 ug) was injected into each mouse via tail vein 3 hr post ATO injection. Images depict 10X magnification obtained from histological sections of FAM-FLIVO stained FSaII tumors. Data courtesy of R.J. Griffin et. al. Technology in Cancer Research 2007. 6(6) 651-654. Window chamber picture from Rob Griffin’s Poster LB209
Kit 980 6 Tests:
• FAM-FLIVO® Poly Caspase Inhibitor (FAM-VAD-FMK), 1 vial, #6218
• 10X Injection Buffer, 5 mL, #6220
• Kit Manual
Kit 981 24 Tests:
• FAM-FLIVO® Poly Caspase Inhibitor (FAM-VAD-FMK), 4 vials, #6218
• 10X Injection Buffer, 5 mL, #6220
• Kit Manual
Mundy-Bosse, BL;Scoville, SD;Chen, L;McConnell, K;Mao, HC;Ahmed, EH;Zorko, N;Harvey, S;Cole, J;Zhang, X;Costinean, S;Croce, CM;Larkin, K;Byrd, JC;Vasu, S;Blum, W;Yu, J;Freud, AG;Caligiuri, MA. MicroRNA-29b mediates altered innate immune development in acute leukemia. J. Clin. Invest. 2016 Dec 1;126(12):4404-4416. doi: 10.1172/JCI85413. Abstract
LaMarche NM, Kane H, Kohlgruber AC, Dong H, Lynch L, Brenner MB. Distinct iNKT Cell Populations Use IFNγ or ER Stress-Induced IL-10 to Control Adipose Tissue Homeostasis. Cell Metab. 2020 Aug 4;32(2):243-258.e6. doi: 10.1016/j.cmet.2020.05.017. Epub 2020 Jun 8. Abstract