FM4-64

• CAT Number: M043414
• CAS Number: 162112-35-8
• Molecular Formula: C30H45Br2N3
• Molecular Weight: 607.51
• Purity: ≥95%
• Synonyms: 3-[4-[(1E,3E,5E)-6-[4-(diethylamino)phenyl]hexa-1,3,5-trienyl]pyridin-1-ium-1-yl]propyl-triethylazanium;dibromide
• InChI: InChI=1S/C30H45N3.2BrH/c1-6-32(7-2)30-20-18-28(19-21-30)16-13-11-12-14-17-29-22-25-31(26-23-29)24-15-27-33(8-3,9-4)10-5;;/h11-14,16-23,25-26H,6-10,15,24,27H2,1-5H3;2*1H/q+2;;/p-2
• InChIKey: AFVSZGYRRUMOFH-UHFFFAOYSA-L
• SMILES: CCN(CC)C1=CC=C(C=C1)C=CC=CC=CC2=CC=[N+](C=C2)CCC[N+](CC)(CC)CC.[Br-].[Br-]

FM4-64 is a very lipophilic, water-soluble styrene dyes, can specifically bind to cell membranes and inner membrane organelles to produce fluorescence. FM4-64 is widely used in endocytic and exospic membrane structure markers.
1. Preparation of FM working solution
1.1 Preparation of the stock solution
Dissolve FM in DMSO to obtain 5 mM of FM.
Note: It is recommended to store the stock solution at -20℃or -80℃away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of FM working solution
Dilute the stock solution in HBSS to obtain 5-20 μM of FM working solution.
Note: Please adjust the concentration of FM working solution according to the actual situation.
2. Cell staining
2.1 Suspension cells (6-well plate)
a. Centrifuge at 1000 g at 4℃for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.The cell density is 1×106/mL
b. Add 1 mL of working solution, and then incubate at room temperature for 5-30 minutes.
c. Centrifuge at 400 g at 4℃for 3-4 minutes and then discard the supernatant.
d. Wash twice with PBS, 5 minutes each time.
e. Resuspend cells with serum-free cell culture medium or PBS.Observation by fluorescence microscopy or flow cytometry.
2.2 Adherent cells
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslip from the medium and aspirate excess medium.
c. Add 100 μL of working solution, gently shake it to completely cover the cells, and then incubate at room temperature for 5-30 minutes.
d. Wash twice with medium, 5 minutes each time. Observation by fluorescence microscopy or flow cytometry.
Storage
-20℃,1 year.
Protect from light.

Reference

[1]. J E Gale, et al. FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel. J Neurosci. 2001 Sep 15;21(18):7013-25.
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[2]. Zal T, et al. Spectral shift of fluorescent dye FM4-64 reveals distinct microenvironment of nuclear envelope in living cells. Traffic. 2006;7(12):1607-1613.
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[3]. Stenovec M, et al. Distinct labelling of fusion events in rat lactotrophs by FM 1-43 and FM 4-64 is associated with conformational differences. Acta Physiol (Oxf). 2007;191(1):35-42.
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[4]. Bolte S, et al, Satiat-Jeunemaitre B. FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc. 2004;214(Pt 2):159-173.
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