Di-8-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 Di-8-ANEPPS storage solution: use ethanol, DMSO or DMF as the storage solution, and the solubility is about 1mg/mL. For example: add 843 μl DMSO to 5 mg Di-8-ANEPPS dye, and use sonication and heating (60°C) to assist dissolution to prepare a stock solution of Di-8-ANEPPS at a concentration of 10 mM.

(2) Preparation of working solution: Dilute the stock solution with an appropriate buffer (such as: serum-free medium, HBSS or PBS) to prepare a working solution with a concentration of 0.5-10 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) 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 1mL of Di-8-ANEPPS working solution (final concentration: 0.2-2uM), incubate at 4°C 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 Adherent Cells

Adherent cells are generally labeled and stained by adding a small amount of the dye stock solution to the medium (0.2-2uM final concentration) and incubating at 4°C for 10 minutes in the dark. After incubation, add PBS to wash 2-3 times, 5 minutes each time.

4. Tissue Sample Staining

The dye concentration used for perfusion labeling tissue samples is 10-50uM, using perfusate to dilute the stock solution. Low labeling temperature (4-20°C) inhibits 

the internalization of the dye. Tissue samples were incubated in probe-containing perfusate for 10-20 min and then washed 3 times with dye-free medium.

Note: The dye incorporation effect of some cell lines is not ideal, and the polysaccharide auxiliary dye can be used to incorporate into the cell membrane. Cells were incubated with polysaccharides and Di-8-ANEPPS dye. Polysaccharides were prepared as a 20% stock solution in DMSO with a working concentration of 0.05%^[1].

5. Retrograde Labeling of Neuronal Cells

Specific neuronal populations can be labeled by localized pressure injection of a high concentration dye solution (10-20 mg/ml in DMSO or ethanol) into the neural tissue^[2].

6. Fluorescence detection: the excitation/emission light of Di-8-ANEPPS is 498/713nm 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.

References:

[1]. Pucihar G, Kotnik T, and Miklavcic D. Measuring the induced membrane voltage with Di-8-ANEPPSJ. Vis. Exp.2009(33)(2016)

[2]. V S Sokolov, et al. Voltage-sensitive styryl dyes as singlet oxygen targets on the surface of bilayer lipid membrane. 2016 Aug;161:162-9.doi:10.1016/j.jphotobiol. 2016. 05. 016.