DiA is a cell membrane green fluorescent dye that diffuses faster in cell membranes than DiO and is often used in cell membrane two-color labeling together with DiI.

DiA is a lipophilic fluorescent stain used to label membranes and other hydrophobic structures. It has environmentally sensitive fluorescence, and although weakly fluorescent in water, the fluorescence is greatly enhanced when bound to membranes or oil-philic biomolecules, such as proteins. Once applied to the cell, this dye diffuses laterally within the plasma membrane of the cell, thereby uniformly staining the entire cell.

DiA can be fixed with paraformaldehyde (no other reagents such as methanol can be used) after dyeing, but it is not recommended to undergo 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). DiA had better staining effect on fixed cells than DiO.

Note:

When DiA is used to stain fixed cell or tissue samples, it is usually fixed with 4% paraformaldehyde formulated in PBS. The use of other inappropriate fixing 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

Usage (for reference only)

1. Dyeing solution preparation

(1) Preparation of storage liquid: storage liquid can be prepared with DMSO or EtOH, the concentration of 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.

Note:

The final concentration of working fluid is recommended to be optimized according to different cell lines and experimental systems.

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.