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574590 Superoxide Anion Detection Kit, Chemiluminescent

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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Luminescence
      Description
      OverviewA sensitive chemiluminescence-based assay kit for the qualitative detection of superoxide anions and superoxide dismutase (SOD) activity in cell cultures. Utilizes a specific enhancement reagent that amplifies the chemiluminescent signal output, and that is non-cytotoxic and non-denaturing to live cells. The principle of this assay is based on the oxidation of luminol by the superoxide anion, which produces photons of light measured with a standard luminometer.
      Store SOD at -20°C and all other components at 4°C.
      Catalogue Number574590
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Supplemented growth medium consisting of
      RPMI 1640 medium with glucose and glutamine
      10% fetal bovine serum
      100 mM sodium pyruvate
      50 U/ml penicillin-streptomycin
      0.1 mM non-essential amino acids
      • Human γ-Interferon (Cat. No. 407306)
      • Dimethyl Sulfoxide (DMSO) (Cat. No. 317275)
      • Phorbol-12-myristate-13-acetate (PMA, TPA) (Cat. No. 524400)
      PMA is an activator of the intracellular enzyme protein kinase C and is a potent stimulator of NADPH oxidase. NADPH oxidase synthesizes superoxide anions. Another stimulator of NADPH oxidase may be substituted for PMA. PMA is a potent carcinogen. Follow standard laboratory safety precautions.
      • Luminometer
      • Polystyrene round-bottom tubes
      References
      ReferencesIrami, K., et al. 1997. Science 275, 1649.
      Stohs, S.J. 1995. J. Basic Clin. Phys. Pharmacol. 6, 205.
      Tanaka, M., et al. 1994. J. Neurochem. 63, 266.
      Wang, J.F., et al. 1991. Biochem. J. 279, 311.
      Lomax, K.J., et al., 1989. Science 245, 409.
      Vilim, V., et al. 1989, Free Rad. Biol. Med. 6, 623.
      Kozumbo, W., et al. 1985. Chem.-Biol. Interact. 54, 199.
      Nakazawa, A., et al. 1985. Biochem. Pharmacol. 34, 481.
      Allred, C., et al. 1980.       Biochem. Biophys. Acta 631, 380.
      Product Information
      Detection methodLuminescence
      Form100 Tests
      FormatRound-bottom polystyrene luminometer tubes
      Kit containsEnhancer Solution, Luminol Solution, Superoxide Anion (SOA) Assay Medium, Superoxide Dismutase (SOD), Xanthine Assay Medium, Xanthine Oxidase, Xanthine Solution, and a user protocol.
      Applications
      Biological Information
      Assay time1.5 h
      Sample TypeCell cultures
      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 Multiple storage requirements
      Storage ConditionsUpon arrival store the Enhancer Solution, Luminol Solution, Xanthine Solution, Xanthine Oxidase, Superoxide Assay Medium, and Xanthine Assay Medium at 4°C and the Superoxide Dismutase at -20°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsEnhancer Solution, Luminol Solution, Superoxide Anion (SOA) Assay Medium, Superoxide Dismutase (SOD), Xanthine Assay Medium, Xanthine Oxidase, Xanthine Solution, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      574590 0

      Documentation

      References

      Reference overview
      Irami, K., et al. 1997. Science 275, 1649.
      Stohs, S.J. 1995. J. Basic Clin. Phys. Pharmacol. 6, 205.
      Tanaka, M., et al. 1994. J. Neurochem. 63, 266.
      Wang, J.F., et al. 1991. Biochem. J. 279, 311.
      Lomax, K.J., et al., 1989. Science 245, 409.
      Vilim, V., et al. 1989, Free Rad. Biol. Med. 6, 623.
      Kozumbo, W., et al. 1985. Chem.-Biol. Interact. 54, 199.
      Nakazawa, A., et al. 1985. Biochem. Pharmacol. 34, 481.
      Allred, C., et al. 1980.       Biochem. Biophys. Acta 631, 380.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      User Protocol

      Revision07-September-2011 JSW
      Form100 Tests
      FormatRound-bottom polystyrene luminometer tubes
      Detection methodLuminescence
      StorageUpon arrival store the Enhancer Solution, Luminol Solution, Xanthine Solution, Xanthine Oxidase, Superoxide Assay Medium, and Xanthine Assay Medium at 4°C and the Superoxide Dismutase at -20°C.
      BackgroundThe superoxide anion (O2-•) is a short-lived radical of molecular oxygen that plays a key role in the immune system and intracellular functions. It is produced by the transfer of an electron to molecular oxygen by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. • O2-• is a potent oxidant released by stimulated leukocytes (monocytes, macrophages, and polymorpho-nuclear leukocytes) that protects against damage infectious organisms. • O2-• is also implicated in oxidative stress damage and tumor promotion. • O2-• has recently been shown to affect cell growth and DNA synthesis through involvement in the Ras cell-signaling pathway. The enzyme superoxide dismutase (SOD) suppresses the activity of the superoxide anion by catalyzing the dismutation of O2-• into O2 and H2O2. Historically, superoxide has been measured using one of three methods: 1. Measuring the uptake of oxygen from the medium (measured by the Clark electrode). 2. Measuring the reduction of exogenously supplied cytochrome c. 3. Measuring luminol-mediated chemiluminescence. The luminol method is the most sensitive; accurate readings can extend three orders of magnitude over the signal range.
      Principles of the assayThe superoxide anion oxidizes luminol in a reaction that produces photons of light that are readily measured with a standard luminometer. The Calbiochem® Superoxide Anion Detection Kit (Cat. No. 574590) utilizes an enhancer that increases the sensitivity of the assay by amplifying the chemiluminescence. Because the enhancer is non-toxic and does not denature components of the subcellular systems, it can be used to assay living cells.
      Materials provided• Enhancer Solutiona (Kit Component No. KP3101): 1 vial, 500 µl, 5 mM
      • Luminol Solutiona (Kit Component No. KP3102): 1 vial, 500 µl, 4 mM
      • Xanthine Solutionb (Kit Component No. KP3103): 1 vial, 100 µl 1 mM
      • Xanthine Oxidaseb (Kit Component No. KP3104): 1 vial, 20 µl, 0.02 U/µl
      • Superoxide Assay Medium (Kit Component No. KP3105): 1 bottle, 25 ml
      • Xanthine Assay Medium (Kit Component No. KP3106): 1 bottle, 25 ml
      • Superoxide Dismutaseb (Kit Component No. KP3107): 1 vial, 20 µl 25 U/µl

      a. This quantity is sufficient for 100 reactions.
      b. This quantity is sufficient for 20 control reactions.
      Materials Required but not provided• Supplemented growth medium consisting of
      RPMI 1640 medium with glucose and glutamine
      10% fetal bovine serum
      100 mM sodium pyruvate
      50 U/ml penicillin-streptomycin
      0.1 mM non-essential amino acids
      • Human γ-Interferon (Cat. No. 407306)
      • Dimethyl Sulfoxide (DMSO) (Cat. No. 317275)
      • Phorbol-12-myristate-13-acetate (PMA, TPA) (Cat. No. 524400)
      PMA is an activator of the intracellular enzyme protein kinase C and is a potent stimulator of NADPH oxidase. NADPH oxidase synthesizes superoxide anions. Another stimulator of NADPH oxidase may be substituted for PMA. PMA is a potent carcinogen. Follow standard laboratory safety precautions.
      • Luminometer
      • Polystyrene round-bottom tubes
      Reagent preparationPreparing the Reagents for the Superoxide Anion Assay PREPARE AND ASSAY ONE REACTION AT A TIME. STORE ALL PROVIDED SOLUTIONS ON ICE WHILE PREPARING AND ASSAYING THE REACTIONS. • Luminal Solution: Dilute 5 µl of 4 mM luminol solution with 89 µl of Superoxide Anion assay medium. Enhancer Solution: Add 5 µl of 5 mM enhancer solution to the mixture of luminol and Superoxide Anion assay medium. PMA: Prepare a 1 mg/ml solution of Phorbol-12-myristate-13-acetate (PMA) in DMSO and vortex it vigorously until the PMA dissolves. To prevent PMA hydrolysis in water, dilute this solution to a concentration of 20 µg/ml PMA with chilled 150 mM NaCl. Vortex the diluted PMA solution for 15 s. The diluted PMA solution should be stored at room temperature and used within 1 h. Add 1 µl of the diluted PMA solution to the mixture of luminol, enhancer, and Superoxide Anion assay medium. This solution is known as the SOA Assay Medium Reagent Mixture. The effective concentration of the enhancer ranges from 0.01 to 2.5 mM. The enhancer may require titration within this range to optimize chemiluminescence. Another stimulator of NADPH oxidase can be substituted for PMA. The concentration of the substituted NADPH oxidase stimulator will require optimization.

      Table 1: SuperoxideAnion (SOA) Assay Medium-Reagent Mixture

      • Control Samples For comparison, unenhanced control samples and control samples without PMA should be assayed along with enhanced samples. The enhanced samples should emit light several fold higher than the control samples. To prepare the unenhanced control samples replace the 5.0 µl of enhancer with 5.0 µl of the Superoxide Anion assay medium. To prepare the control samples without PMA, replace the 1 µl of PMA with 1 µl of Superoxide Anion assay medium. Preparing the Reagents for Superoxide Dismutase PREPARE AND ASSAY ONE REACTION AT A TIME. STORE ALL PROVIDED SOLUTIONS ON ICE WHILE PREPARING AND ASSAYING THE REACTIONS. • Xanthine Oxidase: Add 1 µl of xanthine oxidase and 5 µl of 4.0 mM luminol solution to 94 µl of xanthine assay medium. • Xanthine Solution: Prepare a 50 µM xanthine solution by adding 5.0 µl of the 1.0 mM xanthine solution to 95 µl of xanthine assay medium. Preparing the Reagents for the Xanthine-Xanthine Oxidase Reaction PREPARE AND ASSAY ONE REACTION AT A TIME. STORE ALL PROVIDED SOLUTIONS ON ICE WHILE PREPARING AND ASSAYING THE REACTIONS. • Control Solutions 1. Prepare a xanthine oxidase-luminol-enhancer solution by adding 1 µl of xanthine oxidase, 5 µl of 4.0 mM luminol solution, and 5 µl of 5.0 mM enhancer solution to 89 µl of xanthine assay medium. 2. Prepare a xanthine oxidase - luminol solution by adding 1 µl of xanthine oxidase and 5 µl of 4.0 mM luminol solution to 94 µl of xanthine assay medium. 3. Prepare a xanthine oxidase-enhancer solution by adding 1 µl of xanthine oxidase and 5.0 µl of 5.0 mM enhancer solution to 94 µl of xanthine assay medium. 4. Prepare a xanthine oxidase solution by adding 1 µl of xanthine oxidase to 99 µl of xanthine assay medium. • Xanthine Solution Prepare a 50 mM xanthine solution by adding 5.0 µl of the 1.0 mM xanthine solution to 95 µl of xanthine assay medium.
      Detailed protocolGrowing the tissue culture cells

      1. Seed 25 ml of supplemented growth medium with ~1.25 x 107 cells (resulting in a concentration of ~5 x 105 cells/ml).
      2. Add 500-1000 U (20-40 U/ml) of human γ-interferon to the flask.
      NOTE: If human γ-interferon is not an appropriate differentiator for the cell line of interest, use an appropriate differentiator such as DMSO or retinoic acid (Cat. No. 554720).
      3. Incubate the cells for 2-5 days in a 37°C incubator with 5% carbon dioxide to allow the cells to differentiate. The differentiation process is different for each cell line. To maintain healthy cells, replace the growth medium with fresh supplemented growth medium containing human γ-interferon twice each week.

      Preparing the Tissue Culture Cells

      1. Collect ~5 x 106 cells in a conical tube by centrifuging cell suspension at 3000 x g for 5 min.
      2. Resuspend the cells in 1 ml of fresh supplemented growth medium to increase the reactivity of the cells.
      3. Incubate the cells for 30 min at 37°C.
      4. Aliquot ~5 x 105 cells (100 µl) in a microcentrifuge tube. The minimum number of cells for a successful assay is 1 x 105 cells.
      5. Spin the tube in a microcentrifuge at 1600 rpm for 2 min. Remove and discard the supernatant.
      6. Resuspend the cells in 100 µl of Superoxide Anion assay medium.

      Preparing the Samples for the Superoxide Anion Assay

      Prepare the supernatant of the cells suspected to contain SOD using to an appropriate protocol. The total protein content of the cell supernatant should be 0.8-40.0 µg in a final volume of 5-10 µl. Please refer to the following papers for additional information on the preparation of cell supernatant: Nakano, M., et al. 1990. Anal. Biochem. 187, 277; Somville, M., et al. 1985. Mech. Aging Dev. 29, 35; Nebot, C., et al. 1993. Anal. Biochem. 214, 442.

      Superoxide Anion Assay Protocol

      1. Add 100 µl of the SOA Assay Medium Reagent Mixture to 5.0 x 105 cells suspended in 100 µl of SOA assay medium.

      Table 2: 200 µl Reaction Mixture Concentrations


      2. Incubate the sample for 10-30 min at room temperature. The length of the incubation may need to be optimized. Some types of cells or generators of superoxide anions may require a longer incubation.
      3. Transfer the sample to the bottom of a polystyrene round-bottom tube. Avoid introducing bubbles into the sample during pipetting. Bubbles in the sample may result in inconsistent measurements.
      4. Place the sample in a luminometer and record the light emission at regular intervals.

      Superoxide Dismutase Assay Protocol
      Superoxide dismutase (SOD) catalyzes the dismutation of O2-• into O2 and H2O2. Following this protocol, this kit can be used to assay cell supernatant suspected of containing SOD. The SOD provided with the kit can be used as a positive control. The addition of SOD to the xanthine oxidase-xanthine-luminol reaction results in a reduction in superoxide anion concentration which leads to a decrease in oxidation of luminol and therefore reduced chemiluminescence.

      Note:       Avoid introducing bubbles into the sample during pipetting; bubbles in the sample can result in inconsistent measurements.

      1. Add 100 µl of xanthine oxidase-luminol solution to the bottom of a polystyrene round-bottom tube.
      2. Add 100 µl of the 50 µM xanthine solution to the tube to initiate the reaction.

      Table 3: 200 ml Reaction Mixture Concentrations


      3. Add 5-40 µl of the cell supernatant that is suspected to contain SOD or add 1 µl (25 units) of the provided SOD to the tube.
      4. Place the tube in a luminometer.
      5. Record the light emission at 30-s intervals (from the time the reactants are combined) for up to 2 min.

      Xanthine-Xanthine Oxidase Reaction Protocol (optional Controls)
      The xanthine and xanthine oxidase reaction generates superoxide anions. The reactants xanthine and xanthine oxidase are provided to produce a positive control reaction that demonstrates superoxide anions oxidize luminol.

      Unit Definition: One unit is the amount of xanthine oxidase that converts 1.0 µmol of xanthine to uric acid and forms 2 µmol of superoxide anion per min.

      AVOID INTRODUCING BUBBLES INTO THE SAMPLE DURING PIPETTING. BUBBLES IN THE SAMPLE CAN RESULT IN INCONSISTENT MEASUREMENTS.

      1. Add 100 µl of a control solution (above) to the bottom of a polystyrene round-bottom tube.
      2. Add 100 µl of the 50 µM xanthine solution to the tube of a control solution to initiate the reaction.

      Table 4: 200 ml Reaction Mixture Concentrations


      3. Immediately place the tube in a luminometer.
      4. Record the light emission at 30 s intervals (from the time the reactants are combined) for 2 min.
      5. Repeat steps 1-4 for each control solution.
      Assay characteristics and examples

      Figure 1: Results Expected from the Assay for Superoxide Anion

      The light detected in the assay for superoxide anion activity during a 4-h period. RLU, relative light units; L, luminol; E. enhancer; P, PMA; UD, undifferentiated. The light intensities were 64157, 8065, 15091, 19359, and 22727 RLU for the L+P assay at 0.5, 20, 30, 50, and 100 min, respectively; 2709, 2869, and 2578 RLU for the L+E assay at 0.5, 10, and 160 min, respectively; 2654, 2870, 9071, and 27190 RLU for the UD: L+E+P assay at 0.5, 10, 20, and 50 min, respectively; 90617, 4802, 2869, and 1585 RLU for the UD:L+P assay at 0.5, 10, 30, and 240 min, respectively; and 2709 and 2869 RLU for the UD:L+E assay at 0.5 and 10 min, respectively. Methods. U-937 cells were assayed for superoxide anion activity after incubating for 2-5 days in supplemented growth medium that contained human γ-interferon. After two days, superoxide anion activity was minimal. After three days, the U-937 cells demonstrated superoxide anion activity in response to stimulation with PMA (100 ng/ml).


      Figure 2: Results Expected from the Assay for Superoxide Dismutase

      Light emitted as a result of the xanthine-xanthine oxidase reaction. Superoxide dismutase suppressed the activity of the superoxide anions generated in the reaction. RLU, relative light units; L, luminol; XO, xanthine oxidase; X, xanthine; E, enhancer; SOD, superoxide dismutase. The light intensities were 8413, 4090, and 2223 RLU for the L+XO+X+E+SOD assay and 6222, 4870, 4446, and 3996 RLU for the L+XO+X+SOD assay at 30, 60, 90, and 120 s, respectively.


      Figure 3: Results Expected from the Xanthine-Xanthine Oxidase Reaction

      The light emitted as a result of the xanthine-xanthine oxidase reaction. RLU, relative light units; L, luminol; XO, xanthine oxidase, X, xanthine; E, enhancer. The light intensities were 9284, 5685, and 2829 RLU for the XO+X+E assay and 2222, 1958, and 1853 RLU for the XO+X assay at 30, 60, 90, and 120 s, respectively.
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