DiI (DiIC18(3)) is a lipophilic membrane dye commonly used as a long-term tracer for neurons and other cells. DiI fluoresces very weakly before it enters the cell membrane, and only after it enters the cell membrane can it be excited to emit a strong orange-red fluorescence (λex=549 nm, λem=565 nm).

METHODS: DiI staining:
1. Prepare 1-5 mM of DiI dye stock solution in DMSO or EtOH. Protect from light and store in portions at -20°C. 2.
2. For use, dilute the stock solution with a physiological buffer (e.g., serum-free medium, HBSS, or PBS) to obtain a working solution of 1-5 µM DiI dye. The optimal working concentration must be determined empirically.
3. Suspend cells for staining: Suspend cells at a density of 1×10^6/mL in the dye working solution. Incubate at 37°C for 2-20 min. centrifuge labeled cells at 1000-1500 rpm for 5 min. remove supernatant and resuspend cells in pre-warmed growth medium. Repeat the washing twice.
4. Staining of adherent cells: Cultivate adherent cells on sterile coverslips. Remove the coverslip from the growth medium and gently drain off the excess medium. Pipette 100 μL of staining solution onto one corner of the coverslip and shake gently until all cells are covered. Incubate the coverslips at 37°C for 2-20 min. Drain the dye working solution and wash the coverslips two to three times with growth medium. For each wash, cover the cells with pre-warmed growth medium and incubate for 5-10 min, then drain the medium.
5. The stained cells can be examined by microscopy or flow cytometry.