DiO, or DiOC18(3), is one of the most commonly used cell membrane fluorescent probes, showing green fluorescence. DiO is a lipophilic membrane dye, which can gradually stain the entire cell membrane by lateral diffusion after entering the cell membrane. DiO has very weak fluorescence before entering the cell membrane, but its fluorescence intensity is greatly enhanced when combined with the cell membrane, and it can emit green fluorescence after excitation. Dio has a high quenching constant and excited state lifetime, which can be detected by standard FITC filters.

DiO, as a tracer or long-term tracer, is widely used in forward or reverse, living or fixed nerve cells or tissues. DiO usually does not significantly affect cell viability. In addition to cell membrane fluorescence labeling, DiO can also be used to detect cell fusion and adhesion, cell migration during development or transplantation, detection of lipid diffusion on the cell membrane through FRAP (photodecolorization fluorescence recovery technology), detection of cytotoxicity and labeling of lipoproteins, etc.

DiO can be fixed with paraformaldehyde (no other reagents such as methanol can be used) after dyeing, but it is not recommended to carry out the process of penetration after dyeing. In addition, plasma membrane staining can also be performed well after fixed penetration (penetration with 0.1% TritonX-100 at room temperature). DiO staining strength is generally lower than DiI and is sometimes completely lost in fixed tissue.

Note:

When staining fixed cell or tissue samples with DiO, 4% paraformaldehyde prepared in PBS is usually used for fixation, and the use of other inappropriate fixation fluids will result in a higher fluorescent background.

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

How to use (for reference only)

1. Dyeing solution preparation

(1) Preparation of storage liquid: the storage liquid is prepared with DMSO or EtOH, and the concentration is 1~5 mM.

Note:

a. The unused storage solution is stored at -20℃ to avoid repeated freezing and thawing;

b. When it is found to be difficult to dissolve, ultrasonic treatment or appropriate heating can be used to promote dissolution.

(2) Preparation of working liquid: Dilute the storage liquid with a suitable buffer (such as serum-free medium, HBSS or PBS) and prepare a working liquid with a concentration of 1~30 μM.

2. Suspension cell staining

(1) Adding appropriate volume of dye working fluid to re-suspension cells, making their density 1×10⁶/mL.

(2) Incubate the cells at 37℃ for 2~20 min, and the optimal culture time was different for different cells.

(3) After incubation, centrifuge at 1000-1500 rpm for 5 min. Pour the supernatant and slowly add the growth medium preheated at 37℃ again to resuspend the cells.

(4) Repeat step (3) more than twice.

3. Adherent cell staining

(1) The adherent cells were cultured on a sterile cover slide.

(2) Remove the cover glass from the medium to absorb excess fluid, but keep the surface moist.

(3) Add 100 μL of dye working liquid to one corner of the cover glass, and gently shake the dye to evenly cover all cells.

(4) Cells were incubated at 37℃ for 2~20 min, and the optimal culture time was different for different cells.

(5) Blot the dye working solution, wash the slide with the culture solution for 2 to 3 times, cover all cells with the pre-warmed medium each time, incubate for 5 to 10 min, and then blot the medium. But keep the surface moist.

4. Result detection

The sample can be detected in the medium and can be analyzed by fluorescence microscopy imaging or flow cytometry.