ADHP（Amplex Red） (Synonyms: A 6550, ADHP, Amplex Red)

Storage：

Upon receipt, aliquot and store the ADHP probe and HRP at -20ºC. Avoid multiple freeze/thaw cycles. Store the remaining kit components at 4ºC. ADHP is light sensitive, must be stored accordingly.

Preparation of Reagents

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

• 1X Assay Buffer: 50 mM Tris–HCl buffer pH7.4

• ADHP stock solution：a 10 mM solution in DMSO.

• HRP stock solution: a 100 U/mL solution in glycerol*.

• H₂O₂ stock solution:  an 8.8 M solution

*Note: One unit is defined as the amount of enzyme that will form 1.0 mg purpurogallin from pyrogallol in 20 seconds at pH 6.0 and 20ºC.

• ADHP/HRP Working Solution (Hydrogen Peroxide Assay): If measuring hydrogen peroxide, prepare an ADHP/HRP Working Solution by adding ADHP to a final concentration of 100 µM and HRP to a final concentration of 0.2 U/mL in 1X Assay Buffer (eg. Add 50 µL ADHP stock solution and 10 µL HRP stock solution to 4.940 mL 1X Assay Buffer). This volume is enough for ~100 assays. The ADHP/HRP Working Solution is stable for 1 day. Prepare only enough for immediate use.

• ADHP/H₂O₂ Working Solution (Peroxidase Assay): If measuring peroxidases, prepare the ADHP/H₂O₂ Working Solution by adding ADHP to a final concentration of 100 µM and H₂O₂ to a final concentration of 2 mM in 1X Assay Buffer. First perform a 1:1000 dilution of the stock H₂O₂ in 1X Assay Buffer. Use only enough for immediate applications (eg. Add 5 μL of H₂O₂ to 4.995 mL 1X Assay Buffer). This solution has a concentration of 8.8 mM. Use this 8.8 mM H₂O₂ solution to prepare a 2 mM H₂O₂ solution in ADHP/1X Assay Buffer (eg. Add 50 µL ADHP stock solution and 1.14 mL of the prepared 8.8 mM H₂O₂ solution to 3.81 mL 1X Assay Buffer). This volume is enough for ~100 assays. The Working Solution is stable for 1 day. Prepare only enough for immediate use.

Preparation of Samples

• Cell culture supernatant: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted as necessary. Prepare the H₂O₂ standard curve in the same non-conditioned media. Serum should be avoided, as it interferes with the assay. Note: Maintain pH between 7 and 8 for optimal working conditions as the ADHP is unstable at high pH (>8.5).

• Cell lysate: Resuspend cells at 1-2 x 10⁶ cells/mL in PBS or 1X Assay Buffer. Homogenize or sonicate the cells on ice. Centrifuge to remove debris. Cell lysates can be assayed undiluted or titrated as necessary.

• Plasma, Serum or Urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted as necessary. Undiluted serum or plasma may interfere with the assay.

Notes:

• 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.

• A serial dilution will be necessary depending on the total H₂O₂ or peroxidase present. Extremely high levels of H₂O₂ (≥ 500 µM final concentration) or peroxidase (≥ 100 mU/mL) can lower the fluorescence because excess H₂O₂ or peroxidase can further oxidize the reaction product, Resorufin, to nonfluorescent product Resazurin.

• Samples with NADH concentrations above 10 μM and glutathione concentrations above 50 μM will oxidize the ADHP 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 .

• Avoid samples containing DTT or β-mercaptoethanol since Resorufin is not stable in the presence of thiols (above 10 μM).

Preparation of Standard Curves

• H₂O₂ Standard: To prepare the H₂O₂ standards, first perform a 1:1000 dilution of the stock H₂O₂ in 1X Assay Buffer. Prepare only enough for immediate use (e.g. Add 5 μL of H₂O₂ to 4.995 mL 1X Assay Buffer). This solution has a concentration of 8.8 mM. Use this 8.8 mM H₂O₂ solution to prepare standards in the concentration range of 0 µM – 100 µM by further diluting in 1X Assay Buffer (e.g. Add 11.5 µL of H₂O₂ to 988.5 µL 1X Assay Buffer - see Table 1 below). H₂O₂ diluted solutions and standards should be prepared fresh.

• Peroxidase Standard: To prepare the peroxidase standards, first perform a 1:1000 dilution of the stock HRP in 1X Assay Buffer (e.g. Add 5 μL of HRP stock to 4.995 mL 1X Assay Buffer). Prepare only enough for immediate use. This solution has a concentration of 100 mU/mL. Use this 100 mU/mL solution to prepare standards in the concentration range of 0 mU/mL – 10 mU/mL by further diluting in 1X Assay Buffer (see Table 2 below). HRP diluted solutions and standards should be prepared fresh.

Assay Protocol

I. Hydrogen Peroxide

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 unknown) into an individual microtiter plate well.

3. Add 50 µL of ADHP/HRP Working Solution to each well. Mix the well contents thoroughly and incubate for 30 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.

4. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nm range and for emission in the 590-600 nm range.

5. Calculate the concentration of peroxide within samples by comparing the sample RFU to the standard curve. Subtract the value from the zero H₂O₂ control.

II. Peroxidase

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 (HRP standard, control or unknown) into an individual microtiter plate well.

3. Add 50 µL of ADHP/ H₂O₂ Working Solution to each well. Mix the well contents thoroughly and incubate for 30 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.

4. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nm range and for emission in the 590-600 nm range.

5. Calculate the concentration of peroxidase within samples by comparing the sample RFU to the standard curve. Subtract the value from the zero HRP control.

This protocol only provides a guideline, and should be modified according to your specific needs.