FAM-FLIVO® In vivo Poly Caspase Assay


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.

Catalog # Product Size Quantity Price
980 6 Tests $184.00
981 24 Tests $504.00
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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.

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

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.

FAM-FLIVO in vivo animal rat

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