QIA113 Caspase-8 Detection Kit (FITC-IETD-FMK)

QIA113
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Fluorescence

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      QIA113-1KIT
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          Glass bottle 1 kit
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          Description
          OverviewA fast, convenient, and sensitive method to measure caspase-8 in living cells. The fluorescent marker, FITC-IETD-FMK, is a labeled, cell-permeable, non-toxic inhibitor that binds irreversibly to activated caspase-8 in living cells. Caspase-8 can be detected by the measurement of fluorescence in a fluorescent microplate reader, flow cytometer, or by fluorescence microscopy.
          Catalogue NumberQIA113
          Brand Family Calbiochem®
          References
          Product Information
          Detection methodFluorescence
          Form100 Tests
          FormatFlow cytometry or fluorescence microscopy
          Kit containsLabeled Caspase-8 Inhibitor (FITC-IETD-FMK), Wash Buffer, Unlabeled Caspase Inhibitor (Z-VAD-FMK), and a user protocol.
          Applications
          Biological Information
          Assay time1.5 h
          Sample TypeIntact cells
          Species Reactivity
          • A Broad Range Of Species
          Physicochemical Information
          Emission max.
          Excitation max.
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage -20°C
          Storage ConditionsUpon arrival store the entire contents of the kit at -20°C.
          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 containsLabeled Caspase-8 Inhibitor (FITC-IETD-FMK), Wash Buffer, Unlabeled Caspase Inhibitor (Z-VAD-FMK), and a user protocol.
          Specifications

          Documentation

          Caspase-8 Detection Kit (FITC-IETD-FMK) SDS

          Title

          Safety Data Sheet (SDS) 

          Caspase-8 Detection Kit (FITC-IETD-FMK) Certificates of Analysis

          TitleLot Number
          QIA113

          Brochure

          Title
          The Active Siteル Volume 4, Number 1
          User Protocol

          Revision02-Febuary-2009 RFH
          Form100 Tests
          FormatFlow cytometry or fluorescence microscopy
          Detection methodFluorescence
          Speciesa broad range of species
          StorageUpon arrival store the entire contents of the kit at -20°C.
          Principles of the assayThis Calbiochem® brand Caspase-8 Detection Kit provides a convenient and sensitive means for detecting activated caspase-8 in living cells. The assay employs a Caspase-8 inhibitor conjugated to FITC, the fluorescent marker FITC-IETD-FMK, is a cell-permeable, non-toxic inhibitor that binds irreversibly to activated caspase-8 and allows the detection of Caspase-8 by the measurement of fluorescence intensity (excitation max.: ~485 nm; emission max.: ~535 nm) in a fluorescent microplate reader, by flow cytometry, or by fluorescence microscopy.
          Materials provided• FITC-IETD-FMK (Kit Component No. JA7620-100UL): 1 vial, 100 µl
          • Wash Buffer (Kit Component No. JA7621-100ML): 2 bottles, 100 ml
          • Z-VAD-FMK (Kit Component No. JA7622-10UL): 1 vial, 10 µl
          Detailed protocolCaspase-8 Assay Procedures

          Staining

          1. Induce apoptosis in cells (1 x 106/ml) as desired. Concurrently incubate a control culture without apoptosis induction. An additional negative control can be prepared by adding the caspase inhibitor Z-VAD-FMK at 1 µl/ml to an induced culture to inhibit caspase activity.
          2. Aliquot 300 µl each of the induced and control cultures into eppendorf tubes.
          3. Add 1 µl of FITC-IETD-FMK to each tube and incubate for 0.5-1 h in a 37°C incubator with 5% CO2.
          4. Centrifuge cells at 3,000 rpm for 5 min and remove the supernatant.
          5. Resuspend cells in 0.5 ml of wash buffer and centrifuge again at 3000 rpm for 5 min.
          6. Repeat step 5.

          Proceed depending on method of analysis.

          Quantification by Flow Cytometry

          1. For flow cytometric analysis, resuspend cells in 300 µl of wash buffer and place on ice.
          2. Analyze samples by flow cytometry using the FL-1 channel.

          Detection by Fluorescence Microscopy

          1. Resuspend cells in 100 µl of wash buffer.
          2. Add one drop of the cell suspension to a microslide and cover with a coverslip.
          3. Observe cells under a fluorescence microscope using a FITC filter. Caspase-8 positive cells appear to have brighter green signals, whereas caspase-8 negative control cells show a weaker signal.

          Analysis by Fluorescence Plate Reader

          1. Resuspend cells in 100 µl of wash buffer.
          2. Transfer the cell suspension to each well of the black plate.
          3. Measure the fluorescence intensity at Ex. max. 485 nm and Em. max. 535 nm. For control, use wells containing unlabeled cells.
          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.