1. Preparation of DiI 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 freezing and thawing 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 next item on the agenda is cell suspension

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. The cell density is 1 x 10^6/mL.

2.2. Next, add 1 mL of DiI 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. Adherent cells

3.1. The cells which adhere to the culture should be placed 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 the DiI 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 these samples should be conducted using either fluorescence microscopy or flow cytometry.