Common lipophilic cell membrane dyes DiO, Dil, DiR, Did spectrograms and experimental procedures

Di belongs to long-chain dialkyl carbocyanine dyes. Its unique structure makes it extremely lipophilic and can be combined with lipid-soluble biological structures. At a certain concentration, it can completely dye cell membranes.Di dyes have high quenching coefficients and excited state lifetimes after being excited, and play a great role in the localization and tracing of cells and tissues.

DiD (CAS: 127274-91-3), DiO (CAS: 34215-57-1), DiI (CAS: 41085-99-8), DiR (CAS: 100068-60-8) and DiS dyes areA family of lipophilic fluorescent dyes that can be used to stain cell membranes and other lipid-soluble biological structures.When bound to the cell membrane, its fluorescence intensity is greatly enhanced, and these dyes have high quenching constants and excited-state lifetimes.Once cells are stained, these dyes spread across the cell membrane, and at optimal concentrations can stain the entire cell membrane.Their fluorescence colors are distinct: DiI (orange fluorescence), DiO (green fluorescence), DiD (red fluorescence) and DiR (dark red fluorescence) which allows them to be used for multicolor imaging and flow analysis of live cells.DiI and DiO can be used with standard FITC and TRITC filters, respectively.DiD can be excited by a 633 nm He–Ne laser, has longer excitation and emission wavelengths than DiI, and is more valuable in cell and tissue staining.The infrared fluorescence of DiR penetrates cells and tissues and is used for tracking in in vivo imaging.

Common Di dyes include DiO, DiI, DiD, and DiR, which form fluorescence of different colors after being excited (see Figure 1).

Figure 1. DiO, DiI, DiD, DiR spectra

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■ DiO staining as an example, briefly throw out the in vitro experimental steps

a) Inoculate an appropriate number of cells and incubate at 37°C overnight.

b) Remove the medium, collect the cells by centrifugation, add PBS and wash 2-3 times, 5 minutes each time.

c) Add the corresponding concentration of DiO working solution (5-10 μM) and incubate at room temperature for 30 minutes.

d) Centrifuge at 400 g for 3-4 minutes at 4°C and discard the supernatant.Wash the cells 2-3 times by adding PBS again to fully remove the DiO that did not enter the cells.

f) Cells were resuspended in serum-free medium or PBS and examined under a fluorescence microscope or flow cytometer.

Figure 2. Schematic diagram of the experimental process

■ DiO

DiO is a green fluorescent dye with excitation/emission wavelengths of 483/501 nM, DiO is often used in forward orBacktracking of various types of cells or tissues.(However, compared with the other three dyes, DiO dyes have weaker fluorescence intensity and are slightly less effective for some fixed tissue sections).

Xin Li et al. investigated whether CPCs (cardiac progenitor cell)-derived exosomes (CPCs-Ex) could utilize the mTOR signaling pathway to reduce apoptosis in viral myocarditis.The researchers labelled CPCs-Ex with DiO (Dio can be incorporated into the cell membrane by selective segmentation), and assessed the in vitro uptake of CPCs-Ex by H9C2 cells. Fluorescence microscopy revealed that each cytoplasm of H9C2 cells contained a fluorescent signal (Figure 2).), and reduced apoptosis in H9C2 cells, confirming the anti-apoptotic effect of CPCs-Ex in CVB3-infected cells.

Figure 3. Uptake of DiO-labeled exosomes by H9C2 cells[1]

■ DiI

DiI is a orange-red fluorescent dye with excitation/emission wavelengths of 551/569 nM, DiI was used for forward orFor reverse tracing of various cells or tissues, the fluorescence signal intensity is higher than that of DiO, and its fluorescence signal will be greatly amplified when it enters the cell membrane.

As shown in Figure 4, Dil dye-labeled exosomes were then co-cultured with target cells.Confocal microscopy was used to capture the uptake of Dil-labeled exosomes by HOS cells, where the labeled exosomes emit specific orange-red fluorescence.

Figure 4. Uptake of red fluorescent dye Dil-labeled hBMSC-Exos[2] by HOS and MG63 cells

■ DiD

DiD is a red fluorescent dye with excitation/emission wavelengths of 646/663 nM. DiD has a high fluorescence signal and is not easy toQuenching, low autofluorescence interference, not only for cell/tissue staining, but also most commonly used in small animal live imaging.

Yuxuan Fu et al. developed a drug delivery method to achieve the goal of better entry of anti-COVID-19 drugs into target organs. The researchers used DiD to label the receptor binding domain of extracellular vesicle (EV) viral protein (EV-RBD), and injected into the mouse tail vein, it can be seen by fluorescence imaging that the signal mainly accumulates in the heart, lung, and kidney, and it is worth noting that at 96 h, the fluorescent signal accumulated by the labeled EV-RBD is stillretained in lung tissue, demonstrating the feasibility of the method.

Figure 5. Distribution of DiD-labeled EV-RBD in mice [3]

■ DiR

DiR is a near-infrared dye with excitation/emission wavelengths of 754/778 nm for forward or reverse tracking of various cells or tissues. Like DiD, DiR has a low autofluorescence background and is also used inIn vivo imaging.

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