FLICA 660 Poly Caspase Assay Kit | FLICA assay kit

FLICA 660 Poly Caspase Assay Kit

Cat. Num. AAA52719

• Synonyms: FLICA 660 Poly Caspase; N/A; 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

Application Data

(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 inhibitorreagent.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.)

Application Data

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

Related Product Information for FLICA assay kit

Background: FLICA is a powerful method to assess caspase activity. FLICA probes are cell permeant, noncytotoxic Fluorescent Labeled Inhibitors of CAspases that covalently bind with active caspase enzymes1, 2. AAA Biotech, Inc. has developed a far-red excitation and emission spectra FLICA 660 probe for the detection of cells bearing active caspases. Apoptosis is an evolutionarily conserved process of programmed cell suicide. It is centered on a cascade of proteolytic enzymes called caspases that are triggered in response to pro-apoptotic signals. Like the majority of other proteases, caspases are synthesized as pro-form precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity3. Active caspase enzymes consist of two large (~20 kD) and two small (~10 kD) subunits that non-covalently associate to form a two heterodimer, tetrameric active caspase4, 5, 6. Once activated, caspases cleave protein substrates leading to the eventual disassembly of the cell. Caspases have been identified in organisms ranging from C. elegans to humans. Mammalian caspases play distinct roles in both apoptosis and inflammation. FLICA 660 poly caspase inhibitor probe contains the preferred binding sequence for all caspases, Val-Ala-Asp (VAD). This preferred poly caspase tripeptide binding sequence is labeled at the amino terminus end with a far-red fluorescent 660 dye and linked at the carboxyl end to a fluoromethyl ketone (FMK) reactive entity. The resulting cell permeant, fluorescent molecule, 660-VAD-FMK, optimally excites at 660 nm and emits between 685-690 nm. A conventional red HeNe laser with a 633 nm excitation provides excellent excitation eff iciency, enabling cells labeled with FLICA 660 to be analyzed with most flow cytometers and fluorescence microscopes equipped with electronic grey scale image capabilities. To use FLICA, add it directly to the cell media, incubate, and wash. FLICA is cell-permeant and will eff iciently diff use in and out of all cells. If there is an active caspase enzyme inside the cell, it will covalently bind with FLICA 660-VAD-FMK and retain the far-red fluorescent signal within the cell. Unbound FLICA will diff use out of the cell during the wash steps. Apoptotic cells will retain a higher concentration of FLICA and fluoresce brighter than non-apoptotic cells. There is no interference from pro-caspases or inactive forms of the enzyme. If the treatment is causing cell death via apoptosis, apoptotic cells will have an elevated level of caspase activity relative to non-apoptotic or negative control cells and fluoresce with FLICA. After labeling with FLICA, cells can be counter-stained with other reagents and fixed or frozen.

Product Categories/Family for FLICA assay kit

FLICA 660 Poly Caspase Assay Kit; far-red fluorescence