Novus Caspase Apoptosis Kit, 100 Tests Item ID: NOVNBP229383100Tests

Caspase Apoptosis Kit Summary

Description	FAM Caspase Activity FLICA Apoptosis Detection Kits contain green fluorescent-labeled inhibitor, FAM-VAD-FMK, which is a carboxyfluorescein (FAM) derivative of valylalanylaspartic acid (VAD) fluoromethyl ketone (FMK), a potent inhibitor of caspase activity. When added to a population of cells, the FAM-VAD-FMK FLICA probe enters each cell and covalently binds to a reactive cysteine residue that resides on the large subunit of the active caspase heterodimer, thereby inhibiting further enzymatic activity. Because the FAM-VAD-FMK FLICA reagent becomes covalently coupled to the enzyme, it is retained within the cell, while any unbound FAM-VAD-FMK FLICA reagent will diffuse out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the number of active caspase enzymes that were present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by 96-well-plate based fluorometry, fluorescence microscopy, orflow cytometry. Do not use ethanol-based or methanol-based fixatives to preserve the cells - they will inactivate the FLICA label. Never add the fixative until the staining and final wash steps have been completed.
Immunogen	FLICA Apoptosis Detection Kits use a novel approach to detect active caspases. The methodology is based on a Fluorochrome Inhibitor of Caspases (FLICA). Once inside the cell, the FLICA inhibitor binds covalently to the active caspase 7-11. These inhibitors are cell permeable and non-cytotoxic. For kits using green fluorescence, a carboxyfluorescein-labeled fluoromethyl ketone peptide. inhibitor of caspases is used.
Kit Type	Apoptosis Kit

Packaging, Storage & Formulations

Storage

Store at 4C. Do not freeze.

Applications/Dilutions

Application Notes

Do not use ethanol-based or methanol-based fixatives to preserve the cells - they will inactivate the FLICA label. Never add the fixative until the staining and final wash steps have been completed. Use in fluorophore linked immunosorbent assay reported in scientific literature (PMID: 23958398) Use in In vitro assay reported in scientific literature (PMID 24780216). Use in Immunocytochemistry/immunofluorescence reported in scientific literature (PMID 26975025).

Publications

Read Publications using NBP2-29383 in the following applications: CLIA 1 publication FLOW 4 publications ICC/IF 1 publication In vitro 1 publication

Kit Components

Components

Propidium Iodide Assay Manual FAM-VAD-FMK FLICA Reagent lyophilized Fixative Hoechst Stain 10X Wash Buffer

Reactivity Notes

Rat reactivity reported in scientific literature (PMID: 23958398) Human reactivity reported in scientific literature (PMID: 24780216). Mouse reactivity reported in scientific literature (PMID: 25822986)

Notes

Recommended Materials and Equipment (not all are required): Cultured cells with media Reagents to induce apoptosis 15 mL polystyrene centrifuge tube (1 per sample) Amber vials or polypropylene tubes for storage of 150X concentrate at -20 degrees C, if aliquoted 150 mL or 600 mL graduated cylinder Slides Hemocytometer Clinical centrifuge at 37 degrees C CO2 incubator Vortexer Pipette(s) capable of dispensing at 10uL, 50uL, 200uL, 300uL, 1mL dI H2O, 135 mL or 540 mL needed Phosphate Buffered Saline (PBS) pH 7.4, up to 100 mL needed Dimethyl Sulfoxide (DMSO), 50uL or 200uL needed Ice or 4 degrees C refrigerator to store cells Instrumentation (not all are required): Fluorescence microscope with appropriate filters (excitation 490 nm, emission >520 nm for FLICA; excitation at 490 nm and emission at 635 nm for PI; and if Hoechst is used, a UV-filter with excitation at 365 nm, emission at 480 nm) and slides. Flow cytometer equipped with a 15 mW, 488 nm argon excitation laser, with appropriate filters (excitation 490 nm, emission >520 nm

Background

Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes participate in a series of reactions that are triggered in response to proapoptotic signals and result in the cleavage of protein substrates, causing the disassembly of the cell 1. Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages which lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large (~20 kDa) and two small (~10 kDa) subunits that form two heterodimers which associate in a tetramer 2-4. In common with other proteases, caspases are synthesized as precursors that undergo proteolytic activation, either autocatalytically or in a cascade by enzymes with similar specificity 5. The presence of active caspases is a widely accepted marker of apoptosis. Caspase enzymes specifically recognize a 4 amino acid sequence (on the target substrate) which necessarily includes an aspartic acid residue. This residue is the target for the cleavage reaction, which occurs at the carbonyl end of the aspartic acid residue6.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.