Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*

Protocol summary

1. Prepare cells
2. Incubate cells with MitoROS™ OH580 working solution at 37°C for 60 minutes
3. Incubate cells with test compounds (to induce OH^-)
4. Monitor the fluorescence increase at Ex/Em= 540/590 nm

Important notes
Thaw all the components at room temperature before use.

1. MitoROS™ OH580 stock solution (500X):
Add 50 µL of DMSO (Component C) into the vial of MitoROS™ OH580 (Component A), and mix them well. Note: 25 uL of stock solution is enough for 1 plate. Note: Unused portion can be aliquoted and stored at ≤ -20ºC for more than one month if the tubes are sealed tightly and kept from light. Avoid repeated freeze-thaw cycles.

Add 25 μL of 500X DMSO reconstituted MitoROS™ OH580 stock solution into 10 mL of Assay Buffer (Component B). Mix well. Note: This 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

1. Remove medium, and add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MitoROS™ OH580 working solution into the cell plate. Incubate cells at 37°C for 60 minutes.
   
   
2. To induce hydroxyl radical, treat cells with test compounds in your desired buffer (such as PBS or HHBS) at 37°C for a desired period of time, protected from light. Note: We treated HeLa cells with Fenton reaction (10 µM CuCl₂ and 100 µM H₂O₂) at 37°C for 1 hour to induce exogenous hydroxyl radical. See Figure 1 for details. We treated RAW 264.7 cells with PMA (phorbol 12-myristate 13-acetate) in growth medium at 37°C for 4 hours to stimulate endogenous hydroxyl radical.
   
   
3. Wash cells 2 - 3 times with HHBS or DPBS, and add 100 µL Assay Buffer (Component B) to each well.
   
   
4. Monitor the fluorescence signal in cells using fluorescence microscope with a TRITC filter set, or measure fluorescence increase using fluorescence microplate reader at Ex/Em = 540/590 nm (cut off = 570 nm) with bottom read mode.