Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Optimized for Flow Cytometry*
|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|
|Excitation||488 nm laser|
|Emission||530/30 nm filter|
|Instrument specification(s)||FITC channel|
|Component A: Amplite™ ROS Green||1 vial|
|Component B: Assay Buffer||1 bottle (10 mL)|
|Component C: DMSO||1 vial (200 µL)|
AT A GLANCE
- Prepare cells at the density of 0.5 - 1 × 106 cells/mL
- Add 1 µL 500X Amplite™ ROS Green into 0.5 mL cell suspension
- Stain the cells at 37 ºC for 1 hour
- Treat the cells to induce ROS
- Analyze cells using flow cytometer with FL1 channel (Ex/Em = 490/520 nm)
Thaw all the components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Amplite™ ROS Green stock solution (500X):
Add 100 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: For storage, seal tubes tightly.
For guidelines on cell sample preparation, please visit
SAMPLE EXPERIMENTAL PROTOCOL
- For each sample, prepare cells in 0.5 mL Assay Buffer (Component B) or buffer of your choice at a density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for ROS induction
- Add 1 µL of 500X Amplite™ ROS Green stock solution into 0.5 mL cell suspension.
- Incubate at 37 ºC for 1 hour. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Amplite™ ROS Green. The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment.
- Treat cells by adding 50 µL of 11X test compounds in the desired buffer (such as PBS or HBSS). For control wells (untreated cells), add the corresponding amount of buffer.
- Incubate the cells at 37 ºC for a desired period of time to induce ROS, protected from light. Note: We treated Jurkat cells with 100 µM TBHP (tert-Butyl hydroperoxide) at 37 ºC for 30 minutes to induce ROS. See Figure 1 for details.
- Monitor the fluorescence intensity using a flow cytometer with FL1 channel (Ex/Em = 490/520 nm). Gate on the cells of interest, excluding debris.
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