DiO (Synonyms: DiOC18(3); 3,3'-Dioctadecyloxacarbocyanine perchlorate)

This protocol provides a guide only and should be modified according to your specific needs.

1. DiO cell membrane staining solution

(1). Prepare DMSO or EtOH storage solution: use DMSO or EtOH as the storage solution with a concentration of 1-5mM.

Note: Store the unused stock solution in aliquots at -20°C in the dark and 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 is prepared according to the experiences of 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.

(2). Add 1mL of DiO working solution and incubate at room temperature for 5-30 minutes. The optimal incubation time is different for different cells.

(3). Centrifuge the stained cell test tube at 1000~1500 rpm for 5 minutes.

(4). Centrifuge at 400g for 3-4 minutes at 4°C and discard the supernatant. Pour off the supernatant and add PBS to wash twice, 5 minutes each time.

(5). Resuspend the cells with 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 cover slip from the medium, suck out the excess medium, and place the cover slip 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, and the optimal incubation time is different for different cells.

(5). Discard the dye working solution and wash the coverslip 2~3 times with the culture medium.

4. Microscopic examination: the excitation/emission light of DiO is 484/501nm 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.