FLICA 660 Poly Caspase Assay Kit | FLICA assay kit

FLICA 660 Poly Caspase Assay Kit

Cat. Num. AAA258017

• Synonyms: FLICA 660 Poly Caspase; FLICA 660 Poly Caspase Assay Kit; FLICA assay kit

• Reagents: 660-VAD-FMK
• Sample Protocol: Sample Protocol: FLICA (Fluorescent-Labeled Inhibitor of Caspases) assays offer a simple yet accurate method to measure individual or pan-caspase activity in whole cells. Two sample protocols are outlined below. Detailed instructions are supplied with the kit.
• Suspension Cells: Suspension Cells Culture your cells to a concentration of 2-5 x 10^5 cells/mL. Prepare experimental and control populations. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (may be frozen for future use). Dilute the stock concentrate with 200uL PBS to form the working solution. Add working solution directly to samples and controls at a ratio of 1:30 - 1:60. E.g., add 5-10 uL of working solution to a 300 uL aliquot of suspension cells. Flow cytometry requires less reagent; microscopy applications require more reagent per sample. Incubate 15 - 45 minutes. Wash and spin cells two or three times. If desired, label cells with Hoechst stain, DAPI, or other compatible fluorescent markers. If desired, fix cells with fixative included in kits. Analyze data using a fluorescence microscope or flow cytometer.
• Frozen Tissue: Tissue Sections repare experimental and control populations. Prepare thin frozen tissue sections appropriate to the experiment. Allow sections to thaw before staining with FLICA reagent. Reconstitute each vial of FLICA with 50 uL DMSO to form the stock concentrate. Dilute FLICA stock concentrate 1:50 in PBS to form the tissue section staining solution (TSSS). Use TSSS within 15 minutes of preparation. Add enough TSSS to cover the surface of the tissues and incubate 30 - 60 minutes protected from light. Wash with TBSt, PBSt, or 1X Apoptosis Wash Buffer (twice for 5 min). Set slides in slide incubation dish containing 1X Apoptosis Wash Buffer. Stain nuclei with DAPI or Hoechst, and apply coverslip. Image with appropriate fluorescence microscopy filters. Store samples at 2-8 degree C for short-term storage; staining will last at -20 degree C for long periods.
• Target: Poly Caspases
• Excitation / Emission: 660 nm / 680-690 nm
• Method of Analysis: Flow Cytometer, Fluorescence Microscope
• Types of Samples: Cell Culture, Tissue
• Preparation and Storage: Store at 2-8 degree C

Testing Data

(FLUORESCENCE PROPERTIES OF FLICA 660POLY CASPASE REAGENT:Excitation max:660 nm Emission max: 690nm)

Testing Data #2

(INTRACELLULAR APOPTOSIS DETECTION VIAACTIVE CASPASE ASSAYTo demonstrate thefluorescence propertiesand apoptosis detectioncapability of the newfar-red fluorescent FLICA660 Poly Caspase Assay,Jurkat cells were weretreated with 1 uMstaurosporine for 4 hoursand labeled with FLICA660-VAD-FMK poly caspase inhibitor reagent. Using a Nikon E800 Hyperspectral Microscope equipped with a PhotometricsHQ2 camera, far-red fluorescence emission from apoptotic Jurkat cells wasvacquired with a Cy5 HYQ bandpass filter combination set. FLICA 660-VAD-FMKreagent optimally excites at 660 nm and has an emission max at 690 nm, makingit suitable for use with red lasers on common benchtop flow cytometers andideal for dual staining purposes with green fluorescent probes.)

Related Product Information for FLICA assay kit

Description: Forget the lysates. Assay for apoptosis via caspase activity in whole, living cells with the FLICA 660 Poly Caspase Assay Kit. Ex: 660 nm / Em: 680-690 nm Whole cell analysis via flow cytometry or fluorescence microscopy Flexible multiplexing with additional dyes or probes Compatibility with GFP and green autofluorescence Benchtop flow compatible This in vitro caspase assay employs a new far-red fluorescent caspase inhibitor probe, FLICA 660-VAD-FMK, to label active caspase enzymes in living cells or tissue samples. Image the fluorescent signal using fluorescence microscopy or analyze samples for caspase activity by flow cytometry. The cell permeant FLICA caspase detection probe, 660-VAD-FMK, is comprised of an affinity inhibitor peptide sequence (VAD) and a fluoromethyl ketone (FMK) moiety that enable an irreversible, covalent binding event with active caspase enzymes. This newest FLICA probe is labeled with a far-red fluorescent 660 dye reporter, enabling detection via common fluorescence detection methods.

Background: Assay for Apoptosis via Caspase Activity in Whole, Living Cells (25-50 tests) Apoptosis is an evolutionarily conserved form of cell suicide mediated by a cascade of proteolytic enzymes called caspases. Pro-apoptotic signals activate the enzymatic cascade resulting in the cleavage of protein substrates, leading to the disassembly of the cell. Caspases have been identified in organisms ranging from C. elegans to humans. Members of the mammalian caspase family of cysteinyl aspartate-specific proteases play distinct roles in apoptosis and inflammation. Caspases Active caspase enzymes exhibit catalytic and substrate specificities comprised of short tetra-peptide amino acid sequences that must contain an aspartate in the P1 position. These preferred tetra-peptide sequences have been used to derive peptides that specifically compete for caspase binding. In addition to the distinctive aspartate cleavage site at P1, the catalytic domains of the caspases typically require four amino acids to the left of the cleavage site with P4 as the prominent specificity-determining residue. Addition of a fluoromethyl ketone (FMK) to the tetrapeptide results in an irreversible linkage and permanent inactivation of the cysteine protease enzyme. Furthermore, conjugation of a fluorescent moiety at the amino terminus yields a probe that allows for the detection of caspase activity. FLICA Caspase Assays: Detection Mechanism The red FLICA 660-VAD-FMK caspase detection probe is comprised of the preferred affinity peptide sequence (VAD) targeted by activated pan-caspases, a far-red fluorescent 660 dye label, and a fluoromethyl ketone (FMK) reactive moiety. The resulting fluorescent caspase inhibitor probe forms an irreversible, covalent bond with active caspase enzymes, efficiently labeling the target for detection. Due to its cell permeant nature and fluorescence properties, the far-red FLICA poly caspase probe enables whole cell analysis via common fluorescence detection methods. To use FLICA, add it directly to suspension cell or tissue culture media, incubate, and wash. The cell permeant, far-red FLICA poly caspase probe will efficiently diffuse into cells and irreversibly bind to activated caspase enzymes, thereby retaining the red signal inside caspase-positive cells. Cells not bearing active caspases return to a non-fluorescent status after the wash step. The FLICA 660 poly caspase probe has an optimal excitation at 660 nm and optimal emission range from 680-690 nm. As such, it has demonstrated excellent excitation efficiency with a conventional red HeNe laser with a 633 nm excitation, enabling samples to be analyzed with most flow cytometers and fluorescence microscopes equipped with electronic grey scale image capabilities. Cells labeled with the FLICA reagent may be read immediately or preserved for 16 hours using the fixative. Additional FLICA caspase assays are in development.

Product Categories/Family for FLICA assay kit

FLICA 660 Poly Caspase Assay Kit; far-red fluorescence