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CAS:109244-58-8
Storage:Powder:-20℃，2 years;Insolvent(Mother Liquid):-20℃，6 months;-80℃，1 year
Purity:HPLC≥95%
Appearance:Solid
Dihydrorhodamine 123 (dihydrorhodamine 123, DHR 123), a reduced form of Rhodamine 123, is a commonly used fluorescent mitochondrial dye. Dihydrorhodamine 123 itself is non-fluorescent, it can easily penetrate the cell membrane into the cell and is oxidized by cellular oxidizing substances or REDOX systems to fluorescently Rhodamine 123, which accumulates on the mitochondrial membrane and emits bright green fluorescence (Ex/Em=500/536 nm). Rhodamine 123 cannot be oxidized by nitric oxide (NO), superoxide, or hydrogen peroxide (H2O2) alone, but these reactive oxygen species can oxidize DHR 123 to form Rhodamine 123 when combined with other cellular components such as cytochrome c oxidase or Fe2+. The fluorescence signal can be detected by fluorometer, flow cytometry or fluorescence microscopy.
Dihydrorhodamine 123 can be used to detect reactive oxygen species (ROS), including superoxides (in the presence of peroxidase or cytochrome c) and peroxynitrites. Dihydrorhodamine 123 is widely used in a variety of cells, such as human neutrophils, lung tumor SPC-A-1 cells, endothelial cells, HaCaT cells, eosinophilic cells, mouse mast cells, Guinea pig neutrophils, chondrocytes, and rat proximal renal tubule cells.
Usage (for reference only)
Storage solution preparation
1.7mg Dihydrorhodamine 123 powder was dissolved with 982 μL DMSO to prepare a storage solution with a concentration of 5 mM (1000×). The concentration of the storage solution configuration can be adjusted according to the experimental requirements.
It is recommended that users pack the solution into small quantities and store it at -20℃ according to a single dosage, avoid repeated freezing and thawing, and pay attention to avoid light.
Working fluid preparation
Dilute the storage solution directly with buffer or preheated culture solution to the required working solution concentration (5μM) and mix thoroughly. The specific working liquid concentration should be adjusted and configured according to the experimental needs.
[Note] : During the experiment, the overall dilution ratio should be controlled, and the concentration of DMSO in the culture medium should not exceed 0.1%, so as to avoid the influence of DMSO on cells.
Adherent cell staining
1) Select appropriate cell density to inoculate adherent cells and culture overnight.
2) You can choose interested drug intervention cells and continue to culture for a certain time.
3) Remove the cell culture solution and add DHR 123 working solution for staining.
4) Incubation in a cell incubator at 37℃ for 15-60 minutes.
5) Blot the staining solution, wash the cells with PBS, and observe them with fluorescence microscope.
Suspension cell staining
1) Culture enough suspended cells (1x106 cells/mL).
2) If you want to perform drug stimulation, add drugs of interest to the cells for intervention, and continue to culture for a certain time.
3) Cells were collected centrifugally and washed with PBS.
4) Add DHR 123 working solution to re-suspension cells and incubate at 37℃ for 15 min or longer without light.
5) Flow cytometry was used. Cell suspensions can also be made into slides and examined under a fluorescence microscope.
[Note] : Due to different cell types and experimental systems, the concentration and incubation time of DHR 123 working solution can be adjusted according to pre-experiments or references.
