Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Orange Fluorescence*

Protocol A summary (Fluorescence microplate reader, fluorescence microscope)

1. Prepare cells in growth medium
2. Treat the cells with test compounds to induce ROS
3. Add ROS Brite™ 570 working solution (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
4. Stain the cells at 37 °C for 30 - 60 minutes
5. Monitor the fluorescence increase (bottom read mode) at Ex/Em= 540/570 nm (Cutoff = 550 nm) or fluorescence microscope with TRITC filter set 

Protocol B summary (Flow cytometer)

1. Prepare cells in growth medium
2. Treat cells with test compounds to induce ROS
3. Incubate ROS Brite™ 570 with the cells for 30 - 60 minutes
4. Monitor the fluorescence intensities using flow cytometer with FL2 channel

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. ROS Brite™ 570 stock solution (500X):
Add 40 µL of DMSO (Component C) into the vial of ROS Brite™ 570 (Component A) and mix well to make 500X ROS Brite™ 570 stock solution. Protect from light. Note: 20 µL of 500X ROS Brite™ 570 stock solution is enough for 1 plate. For flow cytometer, 500X ROS Brite™ 570 stock solution can be diluted by 5 folders to 100X in DMSO for convenience. For storage, seal tubes tightly.

Add 20 µL of 500X ROS Brite™ 570 stock solution into 10 mL of Assay Buffer (Component B) and mix well to make ROS Brite™ 570 working solution. Note: This ROS Brite™ 570 working solution is stable for at least 2 hours at room temperature.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

For Protocol A:

1. Treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X 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.
   
   
2. To induce ROS, incubate the cell plate at room temperature or in a 5% CO₂, 37 °C incubator for a desired period of time (for example: 30 minutes treatment for Hela cells with 100 µM tert-butyl hydroperoxide (TBHP)).
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of ROS Brite™ 570 working solution into the cell plate.
   
   
4. Incubate the cells in a 5% CO₂, 37 °C incubator for 30 min to 60 minutes.
   
   
5. Monitor the fluorescence increase with a fluorescence microplate reader (bottom read mode) at Ex/Em = 540/570 nm (Cutoff = 550nm) or observe cells using a fluorescence microscope with TRITC filter set.

For Protocol B:

1. Prepare cells at the density from 5 × 10⁵ to 1 × 10⁶ cells/mL. Note: Each cell line should be evaluated on the individual basis to determine the optimal cell density for apoptosis induction.
   
   
2. Treat cells with test compounds in your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of compound buffer.
   
   
3. To induce ROS, incubate the cell plate at room temperature or in a 5% CO₂, 37 °C incubator for at least 30 minutes or a desired period of time (30 minutes for Hela cells treated with 100 µM tert-butyl hydroperoxide (TBHP)).
   
   
4. Add 1 µL/mL cells of 500X ROS Brite™ 570 stock solution or 5 µL/mL cells of 100X ROS Brite™ 570 stock solution to cells medium.
   
   
5. Incubate the cells in a 5% CO₂, 37 °C incubator for 30 to 60 minutes.
   
   
6. Monitor the fluorescence intensity using a flow cytometer with FL2 channel.