DASPEI, a cationic styrenyl mitochondrial dye, is characterized by a large Stokes shift and excitation/emission wavelengths at 550/573 nm, yielding robust chromogenic properties. It effectively stains mitochondria in live cells and labels presynaptic nerve endings regardless of neuronal activity [1].

Preparation of DASPEI Working Solution: Dissolve DASPEI in DMSO to create a 10 mM stock solution, which should be aliquoted and stored at -20°C or -80°C, protected from light. Dilute the stock with pre-warmed serum-free cell culture medium or PBS to a final concentration of 5-10 μM immediately before use. Adjust the concentration as needed for specific applications.

Cell Staining for Suspension Cells: Collect cells by centrifugation and wash twice with PBS for 5 minutes each, targeting a cell density of 1×10^6/mL. Add 1 mL of DASPEI working solution and incubate at room temperature for 30-60 minutes. Centrifuge at 400 g for 3-4 minutes, discard the supernatant, and wash the pellet twice with PBS for 5 minutes each. Finally, resuspend cells in 1 mL of serum-free medium or PBS for observation with a fluorescence microscope or flow cytometer.

Cell Staining for Adherent Cells: Culture adherent cells on sterile coverslips. After culturing, transfer the coverslip from the medium, remove excess medium, and apply 100 μL of staining working solution. Gently agitate to ensure complete coverage and incubate for 30-60 minutes. Remove the staining solution, wash coverslips with culture medium 2-3 times for 5 minutes each, and observe using a fluorescence microscope.