Amplex Red for H2O2 detection

Amplex Red is a trade name for oxidase-sensitive reagents sold by Invitrogen.  The following protocol applies to the assay kit for hydrogen peroxide (A22188).  Basically, the Amplex Red reagent reacts with H2O2 (in the presence of HRP) to form a red fluorescent molecule called resorufin.  Resorufin can then be quantified in the Tecan by absorbance at 560nm or fluorescence with excitation between 530-560 and emission at 590nm.

Kit Components and stock solutions:

  1. Amplex Red (A, blue) and DMSO (B, green) vials – warm to room temperature before use
  2. Dissolve Amplex Red in 60 μL of DMSO
    1. Each vial is good for ~ 100 samples
  3. Add 4 mL of 5X Reaction Buffer (vial C, white) to 16 mL of DIH2; or 2mL to 8mL
    1. Good for 100 assays of 100 μL each with 10 mL excess for stock solution – likely too much
  4. Dissolve HRP (D, yellow) in 1.0 mL of 1X Reaction Buffer
    1. Unused HRP stock solution should be divided into single-use, 100uL aliquots and stored frozen at -20ºC. These aliquots are stored in Amplex Red Assay Kit container in -20C freezer.
  5. Dilute H2O2 (E, red) into 1X Reaction Buffer to make stock solution
    1. The 3.3% stock = 0.971M; check vial for exact concentration with new kit.
    2. Use solution promptly

Procedure:

  1. Control and exprimental volumes = 50uL, final volumes = 100uL
  2. Make standard curve samples from H2O2 stock & remember 0uM blank; use a 96-well plate and make sure the final volume is 50uL
    1. The assay kit recommends diluting standard curve samples and any H2O2-containing experimental samples 1:1 in 1X reaction buffer since the color-developing reagents are dissolved in 1x reaction buffer (I found similar RFU for H2O2 in reaction buffer and DIH2O).  I wold recommend not diluting experimental samples 1:1 in reaction buffer, though, since this halves your concentration.
    2. High levels H2O2 can produce lower fluorescence than moderately high levels because H2O2 excess can oxidize resorufin to nonfluorescent resazerin
  3. Pipet 50 μL of controls (if any) and experimental samples into individual wells of a 96-well plate; this assay gives you 100 tests, so do duplicates or even triplicates if possible
  4. Make reaction (color-developing) solution by mixing 50 μL of 10 mM Amplex Red reagent stock solution, 100 μL of 10 U/mL HRP stock solution and 4.85 mL of 1X Reaction Buffer
    1. This 5 mL volume is good for ~ 100 assays
  5. Add 50 μL of reaction solution to each 50uL sample
  6. Incubate at room temperature for 30 minutes, protected from light
    1. Fluorescence may be measured multiple times
  7. Use the TECAN to measure absorbance (~560nm) or fluorescence(530nm/590nm excitation/emission); Experiment with gains if needed to give optimal working range