Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Recommended plate||Black wall/clear bottom|
Fluorescence microplate reader
|Recommended plate||Black wall/clear bottom|
|Instrument specification(s)||Bottom read mode|
AT A GLANCE
Protocol summary for Solution Assay
- Prepare and add Amplite™ Green Peroxide Sensor working solution (50 µL)
- Add H2O2 standards or test samples (50 µL)
- Incubate at room temperature for 15 - 60 minutes
- Read fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)
Thaw all the kit components at room temperature before starting the experiment.
Protocol summary for Live Cell Assay
- Prepare cells in growth medium
- Stain cells with Amplite™ Green Peroxide Sensor working solution and incubate for your desired period of time
- Treat cells with test compounds
- Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm) with bottom read mode
Amplite™ Green Peroxide Sensor can be loaded passively into living cells and report the micromolar changes in intracellular H2O2 concentrations. The following is a suggested microscope imaging protocol that can be modified to meet specific research needs.
PREPARATION OF STOCK SOLUTION
1. Amplite™ Green Peroxide Sensor stock solution (250X):
Add 50 µL of DMSO (Component D) into the vial of Amplite™ Green Peroxide Sensor (Component A) to make 250X Amplite™ Green Peroxide Sensor stock solution. Protect from light.
2. H2O2 standard solution (20 mM):
Add 22.7 µL of 3% H2O2 (0.88 M, Component B) into 977 µL of Assay Buffer (Component C) to make 20 mM H2O2 standard solution. Note: The diluted H2O2 standard solution is not stable. The unused portion should be discarded.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11503
Add 50 µL of 20 mM H2O2 standard solution into 950 µL of Assay Buffer (Component C) to get 1000 µM H2O2 standard solution. Take 1000 µM H2O2 standard solution and perform 1:3 serial dilutions to get serially diluted H2O2 standards (HS1 - HS7) with Assay Buffer (Component C).
PREPARATION OF WORKING SOLUTION
Add 20 μL of 250X Amplite™ Green Peroxide Sensor stock solution into 5 mL of Assay Buffer (Component C) to make Amplite™ Green Peroxide Sensor working solution.
For guidelines on cell sample preparation, please visit
SAMPLE EXPERIMENTAL PROTOCOL
Run H2O2 assay in supernatants reaction:
Table 1. Layout of H2O2 standards and test samples in a solid black 96-well microplate. HS= H2O2 Standards (HS1 - HS7, 300 to 0.3 µM); BL=Blank Control; TS=Test Samples
Table 2. Reagent composition for each well.
|HS1 - HS7||50 µL||Serial Dilutions (300 to 0.3 µM)|
|BL||50 µL||Assay Buffer (Component C)|
|TS||50 µL||test sample|
- Prepare H2O2 standards (HS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 50 µL of Amplite™ Green Peroxide Sensor working solution to each well of H2O2 standard, blank control, and test samples to make the total H2O2 assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Amplite™ Green Peroxide Sensor working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 15 to 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 490 ± 10, Emission = 520 ± 10 nm (optimal Ex/Em = 490/525 nm, Cutoff = 515 nm).
Run H2O2 assay in live cells:
- Activate the cells as desired.
- Wash the cells with PBS buffer, incubated the cells with 100 µL/well Amplite™ Green Peroxide Sensor working solution for 5 to 60 minutes or your desired time. For a 384-well plate, add 25 µL/well of Amplite™ Green Peroxide Sensor working solution. Note: For a kinetic measurement, cells can be stained before adding the treatment.
- Monitor the fluorescence increase with a fluorescence plate reader (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) Or image the fluorescence change with a fluorescence microscope using FITC channel.
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