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475866 MitoCapture™ Apoptosis Detection Kit

MitoCapture™ Apoptosis Detection Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorescence
      Description
      OverviewThis kit provides a simple, fluorescent-based method for distinguishing between healthy and apoptotic cells by detecting the changes in the mitochondrial membrane potential. Utilizes MitoCapture™ Reagent, a cationic dye that fluoresces differently in healthy and apoptotic cells. In healthy cells, MitoCapture™ Reagent accumulates and aggregates in the mitochondria, emitting a bright red fluorescence. In apoptotic cells, this reagent cannot aggregate in the mitochondria due to the altered mitochondrial membrane potential, and thus it remains in the cytoplasm in its monomeric form and emits green fluorescence. The fluorescent signals can be easily detected by fluorescent microscopy.
      Catalogue Number475866
      Brand Family Calbiochem®
      References
      Product Information
      Detection methodFluorescence
      Form100 Tests
      FormatFlow cytometry or fluorescence microscopy
      Kit containsMitoCapture™ Reagent, Incubation Buffer, and a user protocol.
      Applications
      Key Applications Flow Cytometry
      Immunofluorescence
      Biological Information
      Assay time30-60 min
      Sample TypeCell suspension or adherent cells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 36/37/38

      Irritating to eyes, respiratory system and skin.
      S PhraseS: 26-36

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      Product Usage Statements
      Intended useThe Calbiochem® MitoCapture™ Apoptosis Detection Kit provides a simple, fluorescent-based method for distinguishing between healthy and apoptotic cells by detecting the changes in the mitochondrial membrane potential.
      Storage and Shipping Information
      Ship Code Ambient Temperature Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsUpon arrival, store the entire contents of the kit at -20°C. Following initial thaw, store the Incubation Buffer at 4°C. Avoid freeze/thaw cycles. Protect from light. Following initial thaw, aliquot the MitoCapture™ Reagent and freeze at -20°C. Avoid freeze/thaw cycles.
      Protect from Light Protect from light
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsMitoCapture™ Reagent, Incubation Buffer, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      475866 0

      Documentation

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      Kit SourceBook - 2nd Edition EURO

      Citations

      Title
    • Rupesh Chaturvedi, et al. (2004) Induction of Polyamine Oxidase 1 by Helicobacter pylori Causes Macrophage Apoptosis by Hydrogen Peroxide Release and Mitochondrial Membrane Depolarization. Journal of Biological Chemistry 279, 40161-40173.
      		•
      User Protocol

      Revision23-July-2010 RFH
      Form100 Tests
      FormatFlow cytometry or fluorescence microscopy
      Detection methodFluorescence
      StorageUpon arrival, store the entire contents of the kit at -20°C. Following initial thaw, store the Incubation Buffer at 4°C. Avoid freeze/thaw cycles. Protect from light. Following initial thaw, aliquot the MitoCapture™ Reagent and freeze at -20°C. Avoid freeze/thaw cycles.
      Intended useThe Calbiochem® MitoCapture™ Apoptosis Detection Kit provides a simple, fluorescent-based method for distinguishing between healthy and apoptotic cells by detecting the changes in the mitochondrial membrane potential.
      Principles of the assayThe MitoCapture™ Apoptosis Detection Kit utilizes MitoCapture™, a cationic dye that exhibits distinct fluorescence in healthy cells versus apoptotic cells. In healthy cells, MitoCapture™ reagent accumulates and aggregates in the mitochondria, giving off a bright red fluorescence. In apoptotic cells, MitoCapture™ reagent cannot aggregate in the mitochondria due to the altered mitochondrial membrane potential, and thus remains in the cytoplasm in its monomer form, generating a green fluroescence. The fluorescent signals can be easily detected by fluorescence microscopy using a band-pass filter (used to detect the fluorescent wavelengths of FITC and rhodamine). These signals can also be analyzed by flow cytometry using the Fluoroscein Isothiocyanate (FITC) channel for green monomers (Ex/Em = 488/530 ± nm) and the Propidium Iodide (PI) channel for red aggregates (Ex/Em = 488/590 ± nm).
      Materials provided• MitoCapture™ Reagent (Kit Component No. KP6701-100UL): 1 bottle, 100 µl
      • Incubation Buffer (Kit Component No. KP6702-100UL): 2 bottles, 100 ml each
      Reagent preparation• Incubation Buffer: aliquot enough Incubation Buffer for the number of assays to be performed (a total of 2 ml for each assay) and pre-warm to 37°C before use. • Diluted MitoCapture™ Reagent: dilute the MitoCapture™ Reagent just prior to use. For each assay, mix 1 µl MitoCapture™ Reagent with 1 ml pre-warmed Incubation Buffer. Vortex to mix. NOTE: The MitoCapture™ Reagent is poorly soluble in aqueous solutions. To remove particulates (optional), centrifuge the dye solution at 13,000 g for 1 min and carefully transfer the supernatant without disturbing pelleted debris.
      Detailed protocolStaining with MitoCapture™ Reagent

      1. Induce apoptosis in cells by desired methods. Prepare a negative control by incubating a separate flask of cells without any treatment.
      2. Following an appropriate incubation time harvest the cells and count. Remove ~1 x 106 cells for each sample and pellet by centrifugation at 500 g for 5 min.
      3. Resuspend the cells in 1 ml Diluted MitoCapture™ Reagent and incubate in a 5% CO2 incubator at 37°C for 15–20 min.
      4. Pellet the cells by centrifugation at 500 g; discard the supernatant.
      5. Resuspend in 1 ml of the pre-warmed incubation buffer.
      6. Analyze by flow cytometry or fluorescent microscopy (see below).

      Detection by Flow Cytometry

      If using flow cytometry, analyze cells immediately following step 5. Mitochondria in healthy cells contain MitoCapture™ aggregates that are detectable using the PI channel (usually FL2). MitoCapture™ monomers in apoptotic cells are detectable using the FITC channel (usually FL1), therefore, the cells generating a green fluorescence represent apoptotic cells.

      Detection by Fluorescence Microscopy

      1. Place the cell suspension from step 5 on a glass slide and cover with a glass coverslip.
      NOTE: For analyzing adherent cells, grow cells on a coverslip and perform the entire procedure directly on this coverslip in a culture dish. Following the incubation in step 3, invert coverslip onto a glass slide.
      2. Analyze the cells immediately under a fluorescence microscope using a band-pass filter (detects fluorescein and rhodamine). MitoCapture™ reagent that has aggregated in the mitochondria of healthy cells generates a red fluorescence. In apoptotic cells, MitoCapture™ reagent cannot accumulate in the mitochondria and remains in the cytoplasm, generating a green fluorescence.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      MitoCapture™ is a trademark of BioVision, Inc.