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.
Human colon carcinoma COLO205 cells were injected SC into female nude mice (tumors circled). After 27 days, animals were treated with a control (animal #1 not shown), TRAIL at 10 mg/kg (animal #2, right), or TRAIL at 25 mg/kg (animal #3 left). Animals were injected with SR-FLIVO™ at 1 and 4 hours to label caspase-positive tumor cells. Images were taken before treatment and at 1, 4, 6, and 24 hours post-treatment. Tumor nodules were excised at 24 hours and imaged (next). The data show that TRAIL induces apoptosis within 1 hour post treatment (a), and the level of apoptosis significantly increases posttreatment (b, c, d). COLO205 tumor cells are being killed; they fluoresce brightly with SR-FLIVO™ compared to no treatment. Data courtesy of Dr. Peter Lassota, Caliper Life Sciences/Xenogen (preliminary data without systems optimization shown).
ACTIVATED CASPASEs IN MOUSE LUNG
Eric Hernady (University of Rochester Medical Center, Rochester, NY) labeled activated caspases in lung parenchymal cells after whole lung irradiation (RT). Mice were given either a 0Gy or 15Gy whole lung dose of gamma irradiation. Six hours post-irradiation, ICT’s SR-FLIVO™ in vivo Apoptosis Kit (cat#983) was injected via mouse tail veins in 100µl boluses. The 1X FLIVO circulated for 18 hours prior to sacrifice. Mice received an intracardiac perfusion with heparinized saline followed by zinc-buffered formalin. Finally, we inflated the lungs prior to removal from the mouse. Tissues were processed, paraffin-embedded and sectioned (5µm). Nuclei were counterstained with DAPI, then lungs were imaged with a fluorescent microscope. Active caspases appear as a bright red cytoplasmic stain with discrete blue nuclei.
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
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“The body weight of the mice was monitored twice weekly. To assess intratumour caspase cleavage, FLIVOTM in vivo apoptosis tracers (Immunochemistry Technologies LLC, MN, United States) were used. The SR FLIVOTM red dye was prepared according to the manufacturer’s protocol and 100 μl was injected into the tail vein of mice after appropriate treatments were completed. After 1 h, whole tumors were excised, digested and analyzed using flow cytometry on the BD FACSAria I.”
Kim JK, Byun MR, Maeng CH, Kim YR, Choi JW. Selective Targeting of Cancer Stem Cells (CSCs) Based on Photodynamic Therapy (PDT) Penetration Depth Inhibits Colon Polyp Formation in Mice. Cancers (Basel). 2020 Jan 14;12(1). pii: E203. doi: 10.3390/cancers12010203. Abstract
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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