FAM-VAD-OPH I in vitro Apoptosis Detection Reagent | FAM-VAD-OPH I detection reagent
FAM-VAD-OPH I in vitro Apoptosis Detection Reagent, Pack of 4 Vials
Synonyms
FAM-VAD-OPH I in vitro Apoptosis; FAM-VAD-OPH I in vitro Apoptosis Detection Reagent; Pack of 4 Vials; FAM-VAD-OPH I detection reagent
Sample Protocol
FAM-OPH Apoptosis Detection Reagents FAM-VAD-OPH I and FAM-VAD-OPH II offer a simple yet accurate method to measure pan-caspase activity in cell culture or live animals. Two sample protocols are outlined below.
Suspension Cells
Culture your cells to a concentration of 2-5 x 10^5 cells/mL. Prepare experimental and control populations; concentrate cells to 5 x 10^5 - 10^6 cells/mL in 300 uL samples. Reconstitute the FAM-OPH reagent with 50 uL DMSO to form the 150X stock concentrate (may be frozen for future use). Dilute the FAM-OPH stock concentrate with 200 uL PBS to form the 30X working solution. Add FAM-OPH working solution directly to samples and controls at a ratio of 1:30. E.g., add 10 uL of working solution to a 300 uL aliquot of suspension cells. Incubate ~1 hour. Wash and spin cells two or three times. If desired, label cells with Hoechst 33342 nuclear stain, DAPI nuclear stain, or other compatible fluorescent markers. If desired, fix cells with formalin-based agents. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.
Frozen Tissue
Prepare animal samples and controls. Reconstitute FAM-OPH with 50 muL DMSO to form the stock concentrate. Dilute the stock concentrate 1:10 by adding 450 muL sterile PBS to form the injection solution. Inject 50-100 muL per animal intravenously. Perform an initial experiment to determine the ideal amount of reagent and time of injection. Let circulate ~1 hour. Excise tissue (or view directly if window chamber option is available). If desired, label with additional stains, such as Hoechst, DAPI, PI, 7-AAD, or an antibody. Tip: Wash excised tissue if necessary to remove excess background stain and improve contrast. If desired, fix cells. Tip: Parafin embedding is not recommended as it may quench green fluorescence. Analyze with a fluorescence microscope, plate reader, or flow cytometer.
Target
Apoptosis, Poly Caspases
Excitation / Emission
488-492 nm / 515-535 nm
Method of Analysis
Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader
Types of Samples
Animal Studies, Cell Culture, Tissue
Preparation and Storage
Store at 2-8 degree C
Related Product Information for FAM-VAD-OPH I detection reagent
Description: Detect apoptosis in cultured cell settings with the green fluorescent poly caspase inhibitor reagent, FAM-VAD-OPH I. FAM-VAD-OPH I: 5-FAM-Val-Ala-Asp(OMe)-2,6-difluorophenoxy-methylketone poly caspase inhibitor; Pack of 4 Vials.
Background: Apoptosis Detection with FAM-VAD-OPH I Poly Caspase Inhibitor Apoptosis is an evolutionarily conserved process of programmed cell suicide mediated by a cascade of proteolytic enzymes called caspases. Once activated in response to pro-apoptotic signals, caspases cleave protein substrates leading to the eventual disassembly of the cell1. Caspase Inhibitors with FMK Reactive Groups Early research studies using short peptide sequences linked to an aldehyde or fluoromethyl ketone (FMK) reactive group identified a number of peptide sequences preferentially targeted by different caspase enzymes. For example, the sequence Asp-Glu-Val-Asp (DEVD) is cleaved by activated caspase-3 and the sequence Val-Ala-Asp (VAD) demonstrates multiple caspase (activated caspase -1, -2, -3, -4, -5, -6, -7 -8, -9) recognition properties2. When linked to an FMK reactive group, the resulting peptide-FMK molecule will form an irreversible, covalent thioether adduct with the reactive SH-site of caspase enzymes. MyBioSource utilized these findings to develop the often-cited FLICA (FLuorescence Labeled Inhibitors of CAspases) and FLIVO (FLuorescence in vIVO, now discontinued) caspase inhibitor and apoptosis detection reagents, both of which have been widely used for years as simple and reliable methods for screening apoptosis in live cells, tissues, and whole animals. These reagents consist of short peptide sequences with varying caspase specificities, a fluorescent label, such as carboxyfluorescein (FAM), and an FMK reactive moiety. The resulting fluorescent, cell-permeant, non-cytotoxic caspase inhibitor reagents can be added to cell culture media for whole cell apoptosis detection. These labeled caspase inhibitors will cross the cell membrane and form irreversible covalent bonds with activated caspase enzymes inside apoptotic cells, which can then be differentiated from non-apoptotic cells by their retained fluorescence. Caspase Inhibitors with O-phenoxy Reactive Groups MyBioSource has now released a novel set of inhibitor reagents that employ an O-phenoxy (OPH) reactive group in place of an FMK group. In a manner analogous to the FMK class of cysteine reactive compounds, the OPH caspase inhibitors form a stable, covalent thioether adduct with the reactive SH-site of caspase enzymes present in apoptotic cells3. OPH inhibitor compounds have the benefit of being extremely non-cytotoxic, display enhanced stability characteristics compared to the FMK analogs, and have been optimized to provide a high level of performance for in vitro applications3-4. The new FAM-OPH line of green fluorescent in vitro and in vivo apoptosis detection reagents includes 5-FAM-Val-Ala-Asp(OMe)-2,6-difluorophenoxy-methyl ketone (FAM-VAD-OPH I), 5-FAM-Val-Ala-Asp(OMe)-Oph (2,6-diF) (FAM-VAD-OPH II), and 5-FAM-Asp-Glu- Val-Asp(OMe)-Oph (2,6-diF) (FAM-DEVD-OPH). A red fluorescent poly caspase inhibitor is also available: SR-VAD-OPH.
Background: Apoptosis Detection with FAM-VAD-OPH I Poly Caspase Inhibitor Apoptosis is an evolutionarily conserved process of programmed cell suicide mediated by a cascade of proteolytic enzymes called caspases. Once activated in response to pro-apoptotic signals, caspases cleave protein substrates leading to the eventual disassembly of the cell1. Caspase Inhibitors with FMK Reactive Groups Early research studies using short peptide sequences linked to an aldehyde or fluoromethyl ketone (FMK) reactive group identified a number of peptide sequences preferentially targeted by different caspase enzymes. For example, the sequence Asp-Glu-Val-Asp (DEVD) is cleaved by activated caspase-3 and the sequence Val-Ala-Asp (VAD) demonstrates multiple caspase (activated caspase -1, -2, -3, -4, -5, -6, -7 -8, -9) recognition properties2. When linked to an FMK reactive group, the resulting peptide-FMK molecule will form an irreversible, covalent thioether adduct with the reactive SH-site of caspase enzymes. MyBioSource utilized these findings to develop the often-cited FLICA (FLuorescence Labeled Inhibitors of CAspases) and FLIVO (FLuorescence in vIVO, now discontinued) caspase inhibitor and apoptosis detection reagents, both of which have been widely used for years as simple and reliable methods for screening apoptosis in live cells, tissues, and whole animals. These reagents consist of short peptide sequences with varying caspase specificities, a fluorescent label, such as carboxyfluorescein (FAM), and an FMK reactive moiety. The resulting fluorescent, cell-permeant, non-cytotoxic caspase inhibitor reagents can be added to cell culture media for whole cell apoptosis detection. These labeled caspase inhibitors will cross the cell membrane and form irreversible covalent bonds with activated caspase enzymes inside apoptotic cells, which can then be differentiated from non-apoptotic cells by their retained fluorescence. Caspase Inhibitors with O-phenoxy Reactive Groups MyBioSource has now released a novel set of inhibitor reagents that employ an O-phenoxy (OPH) reactive group in place of an FMK group. In a manner analogous to the FMK class of cysteine reactive compounds, the OPH caspase inhibitors form a stable, covalent thioether adduct with the reactive SH-site of caspase enzymes present in apoptotic cells3. OPH inhibitor compounds have the benefit of being extremely non-cytotoxic, display enhanced stability characteristics compared to the FMK analogs, and have been optimized to provide a high level of performance for in vitro applications3-4. The new FAM-OPH line of green fluorescent in vitro and in vivo apoptosis detection reagents includes 5-FAM-Val-Ala-Asp(OMe)-2,6-difluorophenoxy-methyl ketone (FAM-VAD-OPH I), 5-FAM-Val-Ala-Asp(OMe)-Oph (2,6-diF) (FAM-VAD-OPH II), and 5-FAM-Asp-Glu- Val-Asp(OMe)-Oph (2,6-diF) (FAM-DEVD-OPH). A red fluorescent poly caspase inhibitor is also available: SR-VAD-OPH.
Product Categories/Family for FAM-VAD-OPH I detection reagent
References
Slee, E. A., et. al. Serial killers: ordering caspase activation events in apoptosis. Cell Death Differ. 6:1067-1074 (1999). Thornberry, N. A., et. al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. J Bio Chem. 272(29):17907-17911 (1997). Chauvier, D., et. al. Broad spectrum caspase inhibitors: from myth to reality? Cell Death Differ. 14:387-391 (2007). Callus, B. A. and Vaux, D. L. Caspase inhibitors:viral, cellular, and chemical. Cell Death Differ. 14:73-78 (2007).
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Product Notes
The FAM-VAD-OPH I (Catalog #AAA258042) is a Detection Reagent and is intended for research purposes only. The product is available for immediate purchase. It is sometimes possible for the material contained within the vial of "FAM-VAD-OPH I in vitro Apoptosis, Detection Reagent" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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