MitoROS Brite™ NIR 780 *Optimized for Detecting Reactive Oxygen Species (ROS) in Mitochnodria*
Example protocol
AT A GLANCE
Before using MitoROS Brite™ NIR 780 for the first time, allow it to thaw at room temperature. Then, briefly centrifuge it to collect the dried pellet.
Prepare the cells in a growth medium.
Stain cells with MitoROS Brite™ NIR 780 working solution.
Treat cells with test compound.
Monitor the fluorescence intensity using a fluorescence microscope equipped with a Cy7 filter set.
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles
Prepare a 5 to 10 mM MitoROS Brite™ NIR 780 stock solution in DMSO. For example, to make a 10 mM stock solution, add 141 μL of DMSO to the MitoROS Brite™ NIR 780 vial.
Note: Any unused stock solution can be stored at -20 °C, protected from light. Avoid repeated freeze-thaw cycles.
PREPARATION OF WORKING SOLUTION
Prepare a working solution of 0.5 to 5 μM by diluting the MitoROS Brite™ NIR 780 stock solution in Hanks solution with 20 mM Hepes buffer (HHBS).
Note: For optimal results, use this solution within a few hours of preparation.
Note: Cover the working solution with foil or store it in a dark place to protect it from light.
SAMPLE EXPERIMENTAL PROTOCOL
Plate the cells as desired in a 96-well black wall-clear bottom plate.
Add 100 µL of the MitoROS Brite™ NIR 780 working solution directly to the cells.
Incubate the cells at 37°C for 15-30 minutes, protected from light.
Note: The concentration and incubation time of MitoROS Brite™ NIR 780 may vary depending on the cell line. Test different concentrations to determine the optimal dose.
Remove the dye working solution and wash the cells twice with HHBS buffer.
Treat cells as desired.
Remove the treatment and was the cells twice with HHBS buffer.
Add HHBS buffer and analyze the cells using a fluorescence microscope equipped with a Cy7 filter set.
Spectrum
References
Authors: Sakurai, Shuhei and Kikuchi, Akito and Gotoh, Hiroaki
Journal: RSC advances (2022): 4094-4100
Authors: Tanev, Mihail and Tomov, Georgi and Karakirova, Yordanka
Journal: Folia medica (2021): 372-376
Authors: Turnell, Biz R and Reinhardt, Klaus
Journal: The journals of gerontology. Series A, Biological sciences and medical sciences (2020): 2278-2285
Authors: Găman, Mihnea-Alexandru and Epîngeac, Mirela Elena and Diaconu, Camelia Cristina and Găman, Amelia Maria
Journal: World journal of diabetes (2020): 193-201
Authors: Phrompet, Chaiwat and Sriwong, Chaval and Srepusharawoot, Pornjuk and Maensiri, Santi and Chindaprasirt, Prinya and Ruttanapun, Chesta
Journal: Heliyon (2019): e01808