Cell Meter™ Fluorimetric Intracellular Total ROS Activity 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
- Prepare cells in growth medium
- Add Amplite™ ROS Green working solution (100 µL/well for a 96- well plate or 25 µL/well for a 384-well plate)
- Stain the cells at 37°C for 60 minutes
- Treat the cells with test compounds to induce ROS
- Monitor the fluorescence increase (bottom read mode) at Ex/Em= 490/525 nm (Cutoff = 515 nm) or fluorescence microscope with FITC filter set
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Amplite™ ROS Green stock solution (500X):
Add 40 µL of DMSO (Component C) into the vial of Amplite™ ROS Green (Component A) and mix well to make 500X Amplite™ ROS Green stock solution. Protect from light. Note: 20 µL of 500X Amplite™ ROS Green stock solution is enough for 1 plate. For storage, seal tubes tightly.
PREPARATION OF WORKING SOLUTION
Add 20 µL of 500X Amplite™ ROS Green stock solution into 10 mL of Assay Buffer (Component B) and mix well to make Amplite™ ROS Green working solution. Note: This Amplite™ ROS Green working solution is stable for at least 2 hours at room temperature.
For guidelines on cell sample preparation, please visit
SAMPLE EXPERIMENTAL PROTOCOL
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Amplite™ ROS Green working solution into the cell plate.
- Incubate the cells in a 5% CO2, 37°C incubator for one hour.
- Treat cells with 20 µL of 11X test compounds (96-well plate) or 10 µL of 6X test compounds (384-well plate) in your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of compound buffer.
- To induce ROS, incubate the cell plate at room temperature or in a 5% CO2, 37°C incubator for at least 15 minutes or a desired period of time (30 minutes for Hela cells treated with 1 mM H2O2).
- Monitor the fluorescence increase with a fluorescence microplate reader (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) or observe cells using a fluorescence microscope with FITC filter set.
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