DiI is a fluorescent probe used to label cell membranes, and DiA, DiI, DiO, DiD, and DiR dyes are a family of lipophilic fluorescent dyes used to label cell membranes and other hydrophobic structures. When incorporated into membranes or combined with lipophilic biomolecules such as proteins, the fluorescence of these environmentally sensitive dyes is greatly enhanced, although they are weakly fluorescent in water. They have a high extinction coefficient, polar-dependent fluorescence and a short excited state lifetime. Once applied to cells, these dyes diffuse laterally within the plasma membrane of the cell, causing the entire cell to stain uniformly at its concentration. The unique fluorescent colors of DiI (orange fluorescence), DiO (green fluorescence), DiD (red fluorescence) and DiR (deep red fluorescence) provide a convenient tool for multicolor imaging and flow cytometry of living cells. DiO and DiI can be used with standard FITC and TRITC filters, respectively.

Di series dye features

DiO dye (green) A long-term tracer of living or stationary cells and tissues. The fluorescence intensity is lower than DiI. The effect of staining on certain fixed tissues is modest.

DiA dye (green) A cell membrane green fluorescent dye that diffuses through cell membranes faster than DiO and is often used with DiI in cell membrane bicolor labeling. Fixation after staining can be performed. DiA had better staining effect on fixed cells than DiO.

DiI dye (orange) Long-term tracer of living or fixed cells and tissues. In addition to labeling cell membranes, it can also detect cell fusion and adhesion, cell migration, etc.

DiB dye (orange) A lipophilic anionic fluorescent dye that detects the potential of cell membranes. It is not inherently fluorescent, but fluoresce when it enters human cells and binds to proteins in the cytoplasm. When it enters the cell, it indicates the increase of intracellular fluorescence intensity, that is, the increase of membrane potential indicates the cell depolarization. Conversely, if the intracellular fluorescence intensity is reduced, that is, the membrane potential is reduced, indicating cell hyperpolarization.

DiD dye (red) staining efficiency is high, uniform, not easy to quench, low cytotoxicity, small background interference.

DiS dye (red) A cell membrane red fluorescent dye that diffuses faster through the cell membrane than DiD and can be fixed after staining. DiS had better staining effect on fixed cells than DiD.

DiR dye (deep red) is often used to label cell membranes, and infrared fluorescence can penetrate cells and tissues for tracing In Vivo imaging. DiR can be fixed after staining.

Selection suggestions: DiI, DiO, DiD and DiR can stain living cells or fixed cells and tissues (please select the appropriate probe according to your needs), DiI is brighter than DiO; DiD, DiR has a longer wavelength and is more suitable for tissue staining

Experimental scheme (for reference only)

Sample analysis

1. Preparation of DiO, DiI DiD, DiS or DiR membrane staining solution:

1.1 Preparation of DMSO or EtOH reserve solution: The reserve solution should be prepared in DMSO or EtOH at 1-5mM.

Note:

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 5uM.

Note:

For different cell types and/or experimental conditions, the concentration of the working solution should be determined empirically.

2. Dye the cells into a suspension:

2.1 The density of suspended cells in the dye working solution was 1×10⁶ / mL.

2.2 Incubate at 37℃ 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℃) 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 100μL 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℃ 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.

Note:

Flow cytometry: Cells labeled with DiO, DiI, DiD, DiS, and DiR can be analyzed using conventional FL1, FL2, FL3, and FL4 flow cytometry detection channels, respectively.