DiSC3(5)

DiSC3(5) is a fluorescent probe commonly used as a tracer dye to evaluate mitochondrial membrane potential. The excitation/emission wavelength of DiSC3(5) is up to 622/670 nm. DiSC3(5) can inhibit the respiratory system associated with mitochondrial NAD, and the IC₅₀ value is 8 μM. DiSC3(5) in the presence of Na⁺/K⁺-ATPase inhibitor ouabain 2 can induce membrane hyperpolarization of Ehrlich ascites tumor cells^[1][2][3]. In Vitro:1. Preparation of DiSC3(5) membrane staining solution:
1.1 Preparation of DMSO or EtOH reserve solution: The reserve solution should be prepared in DMSO or EtOH at 1-5 mM.
Note 1) : The unused portion of the reserve solution should be stored at -20 ° C. Avoid repeated freezing/thawing cycles.
1.2 Prepare the working solution: Dilute the reserve solution into a suitable buffer, such as serum-free medium, HBSS or PBS, to make a working solution of 1 to 5 uM.
Note 2) : For different cell types and/or experimental conditions, the concentration of the working solution should be determined according to experience. It is recommended to test at concentrations at least in excess of the ten-fold range.
2. Dye the cells into a suspension:
2.1 The suspended cell density in the dye working solution was 1×10⁶/ mL.
2.2 Incubate at 37°C for 2-20 minutes. The culture time depends on the cell type. It is incubated first for 20 minutes and then optimized as needed to obtain even labeling.
2.3 Centrifuge the labeled suspension tubes at 1000 to 1500rpm for 5 minutes.
2.4 Remove the supernatant and gently re-suspend the cells in a preheated (37°C) growth medium.
2.5 Wash twice according to steps 2.3 and 2.4.
3. Staining adherent cells:
3.1 The adherent cells were cultured on a sterile glass cover slide.
3.2 Remove the cover glass from the growth medium and gently drain the excess medium. Place the cover glass in the humidity box.
3.3 Transfer the 100 μL dye working solution to the corner of the cover glass and stir gently until all cells are covered.
3.4 Incubate the cover glass at 37°C for 2-20 minutes. The culture time varies according to cell type. Incubation is started for 20 minutes and then optimized as needed to obtain even labeling.
3.5 Drain the dye working solution and clean the cover glass with growth medium two to three times. For each wash cycle, cover the cells with preheated growth medium, incubate for 5-10 minutes, and then drain the medium.
4. Microscope inspection.
5. Flow cytometry detection:
Dis-labeled cells can be analyzed using conventional FL3 flow cytometry detection channels.