DiR, a long-chain carbocyanine dye, is part of the family of carbocyanine dyes extensively used for labeling cells, organelles, liposomes, viruses, and lipoproteins.

Carbocyanine dyes, including DiO (DiOC18(3)), DiI (DiIC18(3)), DiD (DiIC18(5)), DiR, and the dialkyl aminostyryl dye DiA (4-Di-16-ASP), are employed for marking membranes and other hydrophobic structures. These dyes possess high extinction coefficients, display environmental-dependent fluorescence, and exhibit short excited-state lifetimes in lipid environments, becoming fluorescent and photostable when integrated into membranes or attached to lipophilic biomolecules^[1].

DiO, DiI, DiD, and DiR provide green, orange, red, and infrared fluorescence, respectively, enabling multicolor imaging and flow cytometric analysis of live cells. DiO and DiI are compatible with standard FITC and TRITC filters, respectively. DiI and its analogs, known for their minimal cell toxicity, are frequently used for labeling lipoproteins such as LDL and HDL. DiA is commonly utilized in neuronal tracing^[2].

1. Preparation of DiR Reserve Staining Solution

1.1 Preparation of DMSO Reserve Solution: The reserve solution should be prepared in dimethyl sulfoxide (DMSO, concentration 1-5 mM. It should then be applied to the unused solution, which must be aliquoted and refrozen to at least -20°C. It is important to avoid repeated freeze/thaw cycles. The solution can be stored for up to 6 months.)

1.2 Preparation of Working Solution: Please prepare a working solution of 1 to 5 μM by diluting the stock solution into a suitable buffer, such as serum-free medium, HBSS or PBS. It is recommended that the working solution be prepared in advance for use.

Please note that the final concentration of the working solution is dependent on the experimental system. It is therefore recommended that the final concentration conditions be optimised.

2. The cell suspension process

2.1 Centrifuge the cells at 4°C for 3-5 minutes at 1000g, discard the upper layer, and wash with PBS twice for 5 minutes each time.

2.2. Next, add 1 mL of DiR working solution and incubate for 5-30 minutes at room temperature.

2.3. Centrifuge at 400 g for 3-4 minutes at 4°C, then discard the fluid.

2.4. It is recommended that the item be washed twice with PBS for five minutes on each occasion.

2.5. The cells should then be resuspended in serum-free cell culture medium or PBS, and observed using a fluorescence microscope or flow cytometry.

3. The third item on the agenda is the presentation on Adherent cells.

3.1Culture adherent cells on sterile coverslips.

3.2. The coverslip should then be removed from the medium and the excess medium aspirated.

3.3. Next, add 100 μL of DiR working solution, shake gently to ensure full coverage of the cells, and incubate for 5-30 minutes at room temperature.

3.4. Please ensure that the item is washed twice with medium setting for five minutes on each occasion. The observation of this phenomenon can be conducted through the utilisation of fluorescence microscopy or flow cytometry.