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CBA046 Active Caspase-8 Assay Kit

Active Caspase-8 Assay Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
CBA046
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric
      Description
      Overview

      This product has been discontinued.



      Please refer to our complete listing of caspase activity assays for possible alternatives.






      Caspases belong to a family of cysteine proteases that cleave substrates at the carboxyl terminus of asparate residues. Caspase-8 is an initiator caspase responsible for activation of the receptor-mediated pathway of apoptosis. This is a convenient immunofluorescent assay for the quantitative detection of active caspase-8 in cell lysates.
      Catalogue NumberCBA046
      Brand Family Calbiochem®
      Application Data
      *Untreated and treated refer to whether or not compounds were added to the cell culture to induce Caspase-8 activity.
      Materials Required but Not Delivered• Cytochalasin B (Cat No. 250233)
      • Chymostatin (Cat No. 230790)
      • Leupeptin (Cat No. 108975)
      • Antipain (Cat No. 178223)
      • Pepstatin (Cat No. 516481)
      • Phenylmethylsulfonylfluoride (PMSF) (Cat No. 52332)
      • Dimethylsulfoxide (DMSO)
      • Potassium hydroxide (KOH)
      • Polyethylene glycol 8000 (PEG 8000)
      • Pipettes and pipette tips
      • Deionized or distilled water
      • 96-well plates [Costar EIA Plate is recommended]
      • Plate sealers
      • Multi-channel pipette, squirt bottle, manifold dispenser, or automated plate washer
      • 30°C water bath or incubator
      • Centrifuge capable of reaching 12,000 x g
      • Fluorescent plate reader capable of measuring the kinetics of enzymatic reactions at 400 nm excitation and 505 nm emission wavelengths
      • PBS - 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered.
      • Wash Buffer - 0.05% TWEEN®-20 Detergent in PBS, pH 7.2-7.4
      • Block Buffer - 1% BSA (protease-free, Fraction V), 0.05% NaN3, in PBS, pH 7.2-7.4.
      • Assay Buffer - 10 mM DTT, 5% PEG 8000, 0.1% CHAPS, 50 mM HEPES, pH to 7.0 with KOH, 0.2 µm filtered. Prepare fresh daily.
      • Caspase-8 Capture Buffer - 50 mM NaCl, 10 mM DTT, 10% glycerol, 0.1% CHAPS, 50 mM HEPES, pH to 7.0 with KOH. Prepare fresh daily.
      • Caspase-8 Lysis Buffer - 50 mM NaCl, 1 mM DTT, 0.2% CHAPS, 50 mM HEPES, pH to 7.0 with KOH. Prepare fresh daily.
      References
      Product Information
      Detection methodFluorometric
      Form182 Tests
      Format96-well plate
      Kit containsCaspase-8 Capture Antibody, Precursor Caspase-3, DEVD-AFC, and a user protocol.
      Applications
      Biological Information
      Sample TypeCells
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the contents of the kit at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCaspase-8 Capture Antibody, Precursor Caspase-3, DEVD-AFC, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA046 0

      Documentation

      User Protocol

      Revision13-June-2011 RFH
      Form182 Tests
      Format96-well plate
      Detection methodFluorometric
      Specieshuman
      StorageUpon arrival store the contents of the kit at 4°C.
      Principles of the assayThe Calbiochem® Active Caspase-8 Assay Kit captures active caspase-8 using a caspase-8 antibody. Unbound material is removed by washing and inactive precursor caspase-3, a natural substrate for caspase-8, is added. Active caspase-8 cleaves precursor caspase-3, thereby activating caspase-3. The synthetic peptide substrate DEVD-AFC is added and cleaved by caspase-8 activated caspase-3 to generate fluorescent AFC. Fluorescence is measured in a 96-well fluorescent plate reader.
      Materials provided• Caspase-8 Capture Antibody (Kit Component No. JA9143-1EA) 1 vial. 720 µg/ml mouse anti-human Caspase-8 when reconstituted with 200 µl of PBS. After reconstitution, store at 4°C for up to 60 days or aliquot and store at -20°C in a manual defrost freezer or at -70°C for up to 3 months
      • Precursor Caspase-3 (Kit Component No. JA9144-1EA) 1 vial. Reconstitute the Precursor Caspase-3 in 500 µl of Assay Buffer. Aliquot on ice and store at -20°C in a manual defrost freezer or at -70°C for up to 3 months.**Note: To prevent autoactivation, never allow Precursor Caspase-3 to reach temperatures above 4°C prior to the addition to the wells.
      • DEVD-AFC (Kit Component No. JA9145-1EA) 1 vial. 1.3 mg lyophilized substrate. Dissolve in 90 µl DMSO to obtain a 20 mM stock solution. Mix throughly before use. Store at -20°C in a manual defrost freezer or at -70°C for up to 3 months.
      Materials Required but not provided• Cytochalasin B (Cat No. 250233)
      • Chymostatin (Cat No. 230790)
      • Leupeptin (Cat No. 108975)
      • Antipain (Cat No. 178223)
      • Pepstatin (Cat No. 516481)
      • Phenylmethylsulfonylfluoride (PMSF) (Cat No. 52332)
      • Dimethylsulfoxide (DMSO)
      • Potassium hydroxide (KOH)
      • Polyethylene glycol 8000 (PEG 8000)
      • Pipettes and pipette tips
      • Deionized or distilled water
      • 96-well plates [Costar EIA Plate is recommended]
      • Plate sealers
      • Multi-channel pipette, squirt bottle, manifold dispenser, or automated plate washer
      • 30°C water bath or incubator
      • Centrifuge capable of reaching 12,000 x g
      • Fluorescent plate reader capable of measuring the kinetics of enzymatic reactions at 400 nm excitation and 505 nm emission wavelengths
      • PBS - 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered.
      • Wash Buffer - 0.05% TWEEN®-20 Detergent in PBS, pH 7.2-7.4
      • Block Buffer - 1% BSA (protease-free, Fraction V), 0.05% NaN3, in PBS, pH 7.2-7.4.
      • Assay Buffer - 10 mM DTT, 5% PEG 8000, 0.1% CHAPS, 50 mM HEPES, pH to 7.0 with KOH, 0.2 µm filtered. Prepare fresh daily.
      • Caspase-8 Capture Buffer - 50 mM NaCl, 10 mM DTT, 10% glycerol, 0.1% CHAPS, 50 mM HEPES, pH to 7.0 with KOH. Prepare fresh daily.
      • Caspase-8 Lysis Buffer - 50 mM NaCl, 1 mM DTT, 0.2% CHAPS, 50 mM HEPES, pH to 7.0 with KOH. Prepare fresh daily.
      Precautions and recommendations• Keep all solutions and samples on ice while performing the assay.
      • A basic understanding of immunoassay development is required for the successful use of these reagents in immunoassays.
      • A thorough and consistent wash technique is essential for proper assay performance. Wash Buffer should be dispensed forcefully and removed completely from the wells by aspiration or decanting. Remove any remaining Wash Buffer by inverting the plate and blotting it against clean paper towels.
      • Use a fresh reagent reservoir and pipette tips for each step.
      • Solutions containing DTT should be prepared fresh daily.
      • It is recommended that all samples be assayed in duplicate.
      • Avoid microbial contamination of reagents and buffers. This may interfere with the sensitivity of the assay. Buffers containing protein should be made under sterile conditions and stored at 4°C or be prepared fresh daily.
      Preparation• Preparation of Cell Lysates: Immediately before harvesting treated cells, add the following to Caspase-8 Lysis Buffer. Place in an ice bath and keep cold.

      Table 1: Sample Preparation

      1. Centrifuge cells at 1000 x g for 10 min. Note: If adherent cells are used, scrape the cells off the flask prior to centrifugation. Certain treatments may cause adherent cells to lift off the flask. In this case, save the culture media and PBS wash (step 3), centrifuge, and combine these cells with those from step 4. 2. Decant off the culture media, noting the volume. 3. Wash the cells in a volume of PBS equal to the media volume noted in step 2. 4. Centrifuge cells at 1000 x g for 10 min and remove all PBS. 5. Solubilize cells at a minimum cell concentration of 2 x 108 cells/ml in ice cold Caspase-8 Lysis Buffer containing Cytochalasin B and the protease inhibitors listed in the table above. 6. Thoroughly suspend the cells by gently pipetting up and down. Incubate on ice for 10 min. 7. Aliquot 225 µl of the cell lysate into a 2 ml screw-cap microcentrifuge tube and snap freeze in liquid nitrogen. Note: Snap freezing is essential for complete cell lysis. 8. Store lysates at -70°C or thaw at 4°C and use immediately. Stored samples have been used successfully for up to 1 month after preparation. 9. Centrifuge thawed lysates at 12,000 x g at 4°C for 5 min. For assaying, further dilutions may be made in Caspase-8 Capture Buffer. 10. Continue with the Detailed Protocol.
      Detailed protocolKeep all solutions and samples on ice while performing this assay.
      The following controls should be assayed at the same time as the experimental samples. Background levels of caspase-8 and caspase-3 activity and DEVD-AFC cleavage are determined by performing the assay with untreated and treated lysates, with and without precursor caspase-3. See the table below.

      Table 2: Untreated and Treated

      *Untreated and treated refer to whether or not compounds were added to the cell culture to induce Caspase-8 activity.


      1. Dilute the reconstituted Capture Antibody to a working concentration of 4 µg/ml in PBS without carrier protein. Immediately coat a 96-well plate with 100 µl per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
      2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of 3 washes. Wash by filling each well with Wash Buffer (400 µl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
      3. Block plates by adding 300 µl of Block Buffer to each well. Incubate at room temperature for 1-2 h.
      4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
      5. Add 100 µl of cell lysate to the appropriate wells of 96 well plate coated with Capture Antibody. To controls 1 and 2, only add 100 µl of Caspase-8 Capture Buffer; do not add cell lysates.
      6. Cover with a plate sealer and incubate 2 h at room temperature.
      7. Aspirate samples and wash the plate(s) with Wash Buffer, repeating the process two times for a total of 3 washes.
      8. Dilute Precursor Caspase-3 70-fold with ice cold Assay Buffer (145 µl Precursor Caspase-3 added to 10 ml of Assay Buffer per plate). Add 90 µl of diluted Precursor Caspase-3 to each well. To controls 1, 3, and 5, only add 90 µl of Caspase-8 Assay Buffer. Do not add Precursor Caspase-3.
      9. Incubate for 60 min at 30°C. Using a float collar to float the plate in a 30°C water bath is recommended.
      10. Dilute stock 20 mM DEVD-AFC with DMSO to 500 µM (25 µl of 20 mM stock DEVD-AFC and 975 µl of DMSO per plate). Add 10 µl of diluted DEVD-AFC to each well and mix by gently tapping the plate.
      11. Immediately monitor fluorescence at 400 nm excitation and 505 nm emission wavelengths using a fluorescent plate reader capable of measuring the kinetics of enzymatic reactions.
      CalculationsMonitoring the rate of AFC fluorescence generation is highly recommended as this method eliminates the need to subtract background fluorescence from samples. Fluorescence generation is linear for the first 5 min and then the rate decreases slightly. Average the duplicate readings for each control and sample. If using endpoint fluorescence measurements, a sample that contains only DEVD-AFC (Control 1) should be run to determine background fluorescence. DEVD-AFC fluorescence is the major contributor to background and should be subtracted from endpoint fluorescence generated by all samples. Average the duplicate readings for each control and sample, then subtract the DEVD-AFC background.
      SpecificityCaspase-8 is specifically captured in the Active Caspase-8 Assay Kit

      Figure 1: Specificity

      Jurkat cell lysates were treated with 100 ng/ml Fas Ligand for 2 h. Lysates were then incubated in plate wells coated with Caspase-8 Capture Antibody for 2 h. Wells were washed and captured proteins were solubilized in SDS sample buffer. Immunoblots of lysates and captured proteins were performed using an anti-caspase-8 antibody.


      Figure 2: Specificity

      Caspase-8 in treated and untreated Jurkat cell lysates (5 x 105 cells) was captured in wells coated with Caspase-8 Capture Antibody. Wells were washed and precursor caspase-3 was added. After a 60 min incubation at 30°C, cleavage of DEVD-AFC by active caspase-3 was measured using a fluorescent plate reader. Precursor Caspase-3 only (C3 only), untreated lysates (0 h cells), treated lysates (3 h cells), an untreated lysate with Precursor Caspase-3 (0 h cells + C3), and treated lysate with Precursor Caspase-3 (3 h cells + C3) were employed in this assay. Active Caspase-8 and Precursor Caspase-3 are required for DEVD-AFC cleavage.


      Figure 3: Qualification

      Human TK6 T cells, Jurkat T cells, and HEPG2 hepatocytes were treated with Fas Ligand for 2 h and solubilized in Caspase-8 Extraction Buffer at a concentration of 2 x 108 cells/ml. Lysates were analyzed using the Active Caspase-8 Assay Kit. Active Caspase-8 is detected proportional to the amount of cell lysates assayed.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.