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

QIA93
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Overview

Replacement Information

Key Spec Table

Detection Methods
Fluorescence

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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.
      Quality LevelMQ100
      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) MSDS

      Title

      Safety Data Sheet (SDS) 

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

      TitleLot Number
      QIA93

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      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.