QIA93 Caspase-3 Detection Kit (Red-DEVD-FMK)

QIA93
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Fluorescence

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      QIA93-1KIT
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          Description
          OverviewA convenient and sensitive method for the detection of activated caspase-3 in living cells. The fluorescent marker is DEVD-FMK conjugated to sulforhodamine. This cell-permeable, non-toxic inhibitor binds irreversibly to activated caspase-3 in apoptotic cells. The fluorescence intensity can be measured by fluorescence microscopy, fluorescence microplate reader, or flow cytometry.
          Catalogue NumberQIA93
          Brand Family Calbiochem®
          References
          Product Information
          Detection methodFluorescence
          Form100 Tests
          FormatFlow cytometry or fluorescence microscopy
          Kit containsLabeled Caspase-3 Inhibitor (Red-DEVD-FMK), Wash Buffer, Caspase-3 Inhibitor (Z-VAD-FMK), and a user protocol.
          Applications
          Biological Information
          Assay time1.5 h
          Sample TypeCultured 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
          R PhraseR: 36/37/38

          Irritating to eyes, respiratory system and skin.
          S PhraseS: 23-26-36

          Do not breathe fumes.
          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Wear suitable protective clothing.
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage -20°C
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsLabeled Caspase-3 Inhibitor (Red-DEVD-FMK), Wash Buffer, Caspase-3 Inhibitor (Z-VAD-FMK), and a user protocol.
          Specifications

          Documentation

          Caspase-3 Detection Kit (Red-DEVD-FMK) SDS

          Title

          Safety Data Sheet (SDS) 

          Caspase-3 Detection Kit (Red-DEVD-FMK) Certificates of Analysis

          TitleLot Number
          QIA93

          Brochure

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

          Revision07-October-2010 RFH
          Form100 Tests
          FormatFlow cytometry or fluorescence microscopy
          Detection methodFluorescence
          Speciesa broad range of species
          BackgroundThe activation of caspases plays a central role in apoptosis. The Caspase-3 Detection Kit provides a convenient and sensitive means for detecting activated caspase-3 in situ in living cells. The assay utilizes a caspase-3 inhibitor (DEVD-FMK) conjugated to sulfo-rhodamine as the fluorescent in situ marker. Red-DEVD-FMK is cell permeable, nontoxic, and irreversibly binds to activated caspase-3 in apoptotic cells. The red fluorescence label allows for direct detection of activated caspase-3 in apoptotic cells by fluorescence microscopy, flow cytometry, or fluorescence plate reader.
          Materials provided• Red-DEVD-FMK (Kit Component No. JA5900): 1 vial, 100 µl
          • Wash Buffer (Kit Component No. JA5901): 2 bottles, 100 ml
          • Z-VAD-FMK (Kit Component No. JA5902): 1 vial, 10 µl
          Detailed protocolA. Staining Procedure:

          1. Induce apoptosis in cells (1 x 106/ml) by desired method. Concurrently incubate a control culture without induction. An additional control can be prepared by adding the caspase-3 inhibitor Z-VAD-FMK at 1 µl/ml to an induced culture to inhibit caspase-3 activation.
          2. Aliquot 300 µl each of the induced and control cultures into microfuge tubes.
          3. Add 1 µl of Red-DEVD-FMK into each tube and incubate for 0.5-1 h in a 37°C incubator with 5% CO2.
          4. Centrifuge cells at 3000 rpm for 5 min and remove supernatant.
          5. Resuspend cells in 0.5 ml of Wash Buffer and centrifuge again.
          6. Repeat step 5.
          7. Proceed to B, C, or D, depending upon method of analysis.

          B. Quantification by Flow Cytometry

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

          C. Detection by Fluorescence Microscopy

          For fluorescence microscopic analysis, resuspend cells in 100 µl Wash Buffer. Put one drop of the cell suspension onto a microslide and cover with a coverslip. Observe cells under the fluorescence microscope using a rhodamine filter. Caspase positive cells appear to have brighter red signals, whereas caspase negative control cells show much weaker signal.

          D. Analysis by Fluorescence Plate Reader

          For analysis with a fluorescence plate reader, resuspend cells in 100 µl Wash Buffer and transfer the cell suspension to each well of the black plate. Measure the fluorescence intensity at Ex. = ~540 nm and Em. = ~570 nm. For a 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.