DiD perchlorate (Synonyms: D 307, DiIC18(5), NK 3175)

This protocol provides a guide only and should be modified according to 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, the concentration is 1~5mM. For example, take 25mg of DiD (Mw: 959.91g/mol) and dissolve it in 5.21ml of anhydrous DMSO, fully dissolve to obtain a 5mM stock solution.

Note: Store unused stock solutions in aliquots at -20°C, avoid repeated freezing and thawing.

(2) Preparation of working solution: Dilute the stock solution with a suitable 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 needs to be optimized according to different cell lines and experimental systems. It is recommended to start from the recommended concentration and explore the optimal concentration in the range of 10 times.

2. Suspension Cell Staining

(1) The suspended cells were centrifuged at 4°C, 1000-1500rpm for 3-5 minutes, and the supernatant was discarded. Wash twice with PBS for 5 minutes each.

(2) Add 1 mL of DiD perchlorate working solution and incubate at room temperature for 5-30 minutes.

Note: Different cells have different optimal culture time, 20min can be used as the initial incubation time, and then optimized to ensure uniform labeling results.

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

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

3. Staining of Adhesive Cells

(1) Culture adherent cells on sterile coverslips.

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

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

(4) Incubate at room temperature for 5-30 minutes. Different cells have different optimal culture time, 20min can be used as the initial incubation time, and then optimized to ensure uniform labeling results.

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

4. Microscopic examination: the excitation/emission light of DiD perchlorate is 650/670nm respectively.

Note:

1) Fluorescent dyes have quenching problems, please try to avoid light to slow down fluorescence quenching.

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