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 red 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.
SR-FLIVO® In vivo Poly Caspase Assay
$184.00 – $504.00
FLIVO® (FLuorescence in vIVO) is a powerful method for assessing caspase activity in vivo. SR-FLIVO poly caspase probes are non-cytotoxic, cell-permeant fluorescent inhibitors of caspases optimized for use in whole live animals. ICT’s SR-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 sulforhodamine B (SR) dye and a fluoromethyl ketone (FMK) reactive entity.
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1. Prepare samples and controls.
2. Dilute 10X Injection Buffer 1:10 with 45 mL diH20.
3. Reconstitute SR-FLIVO with 50 µL DMSO.
4. Dilute SR-FLIVO 1:12 with 550 µL 1X Injection Buffer.
5. Inject 100 µL intravenously.
6. Let SR-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. SR-FLIVO excites at 550-580 nm and emits at 590-600 nm.
• Reagent Name: SR-FLIVO® Poly Caspase Inhibitor (SR-VAD-FMK)
• Target: Poly Caspase
• Excitation/Emission: 550-580 nm/590-600 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
IN VIVO APOPTOSIS DETECTION
SR-FLIVO was used to assess the effectiveness of arsenic trioxide (ATO) treatment on SCK mammary tumors in vivo. Using a window chamber to view tumors directly, SCK mammary tumor cells were grown under the skin of A/J mice for 7 days. Test mice were treated with ATO and the control mice received a placebo (24 hours). All mice were injected IV with SR-FLIVO and images were captured 30 minutes later. The control SCK tumor (A.) exhibited a base level of apoptosis as expected (18%, FACS data not shown), while the ATO-treated tumor (B.) exhibited a much higher level of apoptosis (36%, FACS data not shown). Bright spots indicate high levels of caspase activity within the tumor. ATO treatment doubled the level of caspase activity. Data courtesy of Dr. Robert Griffin, University of Minnesota.
Kit 982 6 Tests:
• SR-FLIVO® Poly Caspase Inhibitor (SR-VAD-FMK), 1 vial, #6219
• 10X Injection Buffer, 5 mL, #6220
• Kit Manual
Kit 983 24 Tests:
• SR-FLIVO® Poly Caspase Inhibitor (SR-VAD-FMK), 4 vials, #6219
• 10X Injection Buffer, 5 mL, #6220
• Kit Manual
Kuchay, S;Giorgi, C;Simoneschi, D;Pagan, J;Missiroli, S;Saraf, A;Florens, L;Washburn, MP;Collazo-Lorduy, A;Castillo-Martin, M;Cordon-Cardo, C;Sebti, SM;Pinton, P;Pagano, M. PTEN counteracts FBXL2 to promote IP3R3- and Ca(2+)-mediated apoptosis limiting tumour growth. Nature. 2017 June 22. doi: 10.1038/nature22965. https://www.nature.com/nature/journal/vaop/ncurrent/full/nature22965.html.
“For the analysis of apoptosis in tumour tissue sections, after a retro orbital injection of 100µl of SR-FLIVO probe (Immunochemistry Technology), the reagent was allowed to circulate in mice for 30min.”
Missiroli, S;Bonora, M;Patergnani, S;Poletti, F;Perrone, M;Gafà, R;Magri, E;Raimondi, A;Lanza, G;Tacchetti, C;Kroemer, G;Pandolfi, PP;Pinton, P;Giorgi, C. PML at Mitochondria-Associated Membranes Is Critical for the Repression of Autophagy and Cancer Development. Cell Report. 2016 August 30, Pages 2415-2427. doi:10.1016/j.celrep.2016.07.082. http://www.sciencedirect.com/science/article/pii/S2211124716310282. Full Article
“After an intravenous (i.v.) injection of 100 μl SR-FLIVO (Immunochemistry Technologies) via the lateral tail vein, the FLIVO reagent was allowed to circulate in the mouse for 30 min before sacrifice.”