DiI (DiIC18(3))

This plan only provides a guide, please modify it to meet your specific needs.1. Preparation of cell membrane staining solution

(1) Prepare DMSO or EtOH storage solution: The storage solution is prepared with DMSO or EtOH, with a concentration of 1~5mM.

Note: Unused storage solution should be stored in aliquots at -20°C in the dark and avoid repeated freezing and thawing.

(2) Preparation of working solution: Dilute the storage solution with an appropriate buffer (such as serum-free medium, HBSS or PBS) to prepare a working solution with a concentration of 0.5~5 μM.

Note: The final concentration of the working solution is prepared based on experience with different cells and experiments. Optimum conditions can be found from more than ten times the recommended concentration.

2. Suspension cell staining

(1) Centrifuge the suspended cells at 1000g for 3-5 minutes at 4°C and discard the supernatant. Wash twice with PBS for 5 minutes each time.

(2) Add 1 mL of dye working solution and incubate at room temperature in the dark for 5-30 minutes.

Note: The optimal culture time for different cells is different and can be adjusted according to specific experimental needs.

(3) After the incubation, centrifuge at 1000-1500g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.

(4) Resuspend the cells in pre-warmed serum-free cell culture medium or PBS. Observe by fluorescence microscopy or flow cytometry.

3. Staining of adherent cells

(1) Culture adherent cells on sterile coverslips.

(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment.

(3) Add 100uL of dye working solution from one corner of the coverslip and shake gently to evenly cover all cells with the dye.

(4) Incubate at room temperature in the dark for 5-30 minutes. The optimal culture time for different cells is different.

(5) After the incubation, discard the dye working solution and wash the coverslip 2 to 3 times with pre-warmed culture solution.

4. Exosome staining

(1) Take an appropriate volume of Di dye stock solution and add it to the collected exosome filtration supernatant to reach a final concentration of 5 μM, and mix gently using a pipette;

(2) Incubate at room temperature in the dark for 30 minutes, turning gently every 5 minutes.

(3) Transfer the incubated sample to a centrifuge tube and centrifuge it in an ultracentrifuge at 100000g and 4°C for 30 minutes;

(4) After centrifugation, carefully tilt and discard the supernatant in a clean bench. Use a micropipette with a sterile tip to remove the liquid that forms in the test tube but does not drip. This is the marked outer layer. secretion body;

Note: During this step, the free dye disappears with the decanted supernatant, so the solution can be decanted, but always be careful not to lose the precipitate.

5. Microscope detection: The excitation/emission light of DiI (DiIC18(3)) is 549/565 nm respectively.

Precautions:

1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.

2) For your safety and health, please wear a lab coat and disposable gloves.

References:

[1]. Mufson, E.J., Brady, D.R., and Kordower, J.H.Tracing neuronal connections in postmortem human hippocampal complex with the carbocyanine dye DiINeurobiol. Aging11(6)649-653(1990).

[2]. John Santelices,et. Fluorescent Labeling of Small Extracellular Vesicles (EVs) Isolated from Conditioned Media. 2022 Jun 20;12(12):e4447. doi: 10.21769/BioProtoc.4447.