SynaptoRedTM C2 (Synonyms: FM 4-64,FM1-43,SynaptoRedC2,Lipophilic probe,)

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

1. Preparation of SynaptoRedTM C2 (FM4-64) 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 the unused stock solution in aliquots at -20°C or -80°C in the dark and avoid repeated freezing and thawing.

(2) Working solution preparation: Dilute the stock solution with an appropriate buffer (such as: serum-free medium, HBSS or PBS) to prepare a 5-20 μM FM working solution.

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) 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 SynaptoRedTM C2 (FM4-64) 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. Microscope detection: the excitation/emission light of SynaptoRedTM C2 (FM4-64) is 750/780nm 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.