Di-4-ANEPPS

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.

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 0.5-10μM.

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) 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 Di-4-ANEPPS working solution and incubate at room temperature for 5-30 minutes. The optimal incubation time is different for different cells.

(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, and the optimal incubation time is different for different cells.

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

4. Microscopic examination: the excitation/emission light of Di-4-ANEPPS are 495/705nm respectively.

Note:

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

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