OxiSelect™ Monoamine Oxidase Assay Kit (Colorimetric)

1. 96-well Protein Binding Plate : One 96-well strip plate
2. 200X Colorimetric Probe : One 55 μL vial
3. HRP : One 100 μL tube of 100 U/mL solution in glycerol
4. Hydrogen Peroxide : One 100 μL amber tube of an 8.82 M solution
5. 100X Tyramine : One 100 μL amber tube (substrate for MAO-A, MAO-B, and SSAO)
6. 100X Benzylamine : One 100 μL amber tube (substrate for MAO-B and SSAO)
7. MAO-A Inhibitor : One 50 μL amber tube of 20 mM Clorgyline solution
8. MAO-B Inhibitor : One 50 μL amber tube of 20 mM Pargyline solution
9. 10X Assay Buffer : One 25 mL bottle

• Detects MAO-A and MAO-B in biological samples
• MAO substrates and inhibitors included
• Hydrogen peroxide standard included
• Available with fluorometric or colorimetric detection

Note: All reagents must be brought to room temperature prior to use. 3

• 1X Assay Buffer: Dilute the stock 10X Assay Buffer 1:10 with deionized water for a 1X solution. Stir or vortex to homogeneity.
• MAO-A Inhibitor: Immediately before use, prepare a 100 μM solution in 1X PBS (e.g. Add 5 μL of the 20 mM inhibitor to 0.995 mL 1X PBS). Vortex thoroughly. Store solutions at -20 °C.
• MAO-B Inhibitor: Immediately before use, prepare a 100 μM solution in 1X PBS (e.g. Add 5 μL of the 20 mM inhibitor to 0.995 mL 1X PBS). Vortex thoroughly. Store solutions at -20 °C.
• Assay Working Solution: Immediately before use, prepare an Assay Working Solution using Table 1 below as a guide based on the number of assays needed. Prepare by diluting the 200X Colorimetric Probe 1:100, 100X Tyramine or 100X Benzylamine 1:50, and 100 U/mL HRP to a final concentration of 0.2 U/mL in 1X Assay Buffer. The Assay Working Solution should be protected from light and used within 2 hours. Prepare only enough for immediate use. Note: The Assay Working Solution will appear slightly pink in color. This is normal background and should be subtracted from all absorbance values. 1X Assay 100X Tyramine HRP 200X Total Volume Number of Assays Buffer (mL) or 100X (μL) Colorimetric of Assay in 96-well Plate Benzylamine Probe (μL) Working (50 μL/well) (μL) Solution (mL) 4.840 100 10 50 5 100 2.420 50 5 25 2.5 50 0.968 20 2 10 1 20 Table 1. Preparation of Assay Working Solution.

Note: Samples should be assayed immediately or stored at -80 °C prior to performing the assay. Optimal experimental conditions for samples must be determined by the investigator. The following recommendations are only guidelines and may be altered to optimize or complement the user's experimental design. A set of serial dilutions is recommended for samples to achieve optimal assay results and minimize possible interfering compounds. Run proper controls as necessary. Always run a standard curve with samples.

• Platelets: Prepare freshly drawn blood into polypropylene or polyethylene tubes with 1/10 volume of 1.5 % EDTA/0.7 % NaCl (e.g. 20 mL blood, 2 mL buffer). The volume of blood depends on the experimental needs. Centrifuge the tubes at 200 x g for 15 minutes at 4 °C. Carefully transfer the supernatant containing platelet rich plasma into clean tubes. Avoid carry over of erythrocytes. Centrifuge at 2000 x g for 10 minutes at 4 °C. Store the platelet pellet at -80 °C until use. Immediately before testing, prepare cell lysate by sonication of the pellet in 1X PBS (Youdim, Ref. 4).
• Cell or tissue mitochondrial fractions: Isolate mitochondria using differential centrifugation for cell or tissue samples, or by the method of choice. Mitochondrial samples can be diluted in 1X PBS. Notes: 4
• All samples should be assayed immediately or stored at -80 °C for up to 1-2 months. Run proper controls as necessary. Optimal experimental conditions for samples must be determined by the investigator. Always run a standard curve with samples.
• Samples with NADH concentrations above 10 μM and glutathione concentrations above 50 μM will oxidize the probe and could result in erroneous readings. To minimize this interference, it is recommended that superoxide dismutase (SOD) be added to the reaction at a final concentration of 40 U/mL (Tatyana et al, Ref. 2).
• Avoid samples containing DTT or β-mercaptoethanol since the probe is not stable in the presense of thiols (above 10 μM).
• Maintain pH between 7 and 8 for optimal working conditions as the probe is unstable at high pH (>8.5).

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate.
2. Add 50 μL of each sample (H2O2 standard, control or sample) into an individual microtiter plate well.
3. If assaying with MAO inhibitors, add 5 μL of the 100 μM inhibitor to the appropriate MAO sample wells. Add 5 μL Assay Buffer to the H2O2 standards and samples without inhibitor. Mix the well contents thoroughly by pipetting or on a horizontal shaker and incubate 30 minutes at room temperature to allow the inhibitor to react with the enzyme. Note: The concentration of MAO-A or MAO-B inhibitors may be adjusted by the user. 5
4. Add 50 μL of Assay Working Solution to each well. Mix the well contents thoroughly and incubate for 45-60 minutes at room temperature protected from light. Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the kinetics of the reactions.
5. Read the plate with a spectrophotometric microplate reader in the 540-570 nm range.

1. Calculate the average absorbance values for every standard, control, and sample. Subtract the average zero standard value from itself and all standard and sample values. This is the corrected background absorbance. If sample background control value is high, subtract the sample background control value from the sample reading.
2. Plot the corrected absorbance for the H2O2 standards against the final concentration of the hydrogen peroxide standards from Table 2 to determine the best slope (μM-1). See Figure 1 for an example standard curve.
3. Use the standard curve to determine the hydrogen peroxide concentration generated by MAO.
4. Determine the monoamine oxidase enzyme activity of the samples using the equation below. Substitute the corrected fluorescence values for each sample. Remember to account for dilution factors. [H2O2] generated MAO Activity (Units/L) = x Sample dilution Reaction Time (minutes) Note: One unit of MAO catalyzes the formation of 1 μmole of hydrogen peroxide per minute at 25 °C. 7