Fluorescent dye for binding to and staining of cell membranes

1. Preparation of FM working solution
1.1 Preparation of stock solution
Prepare 5 mM stock solution with DMSO.
1.2 Preparation of working solution
Prepare 5-20 μ M FM4-64 working solution using preheated HBSS solution.
Note: Please adjust the concentration of FM4-64 working solution according to your specific needs, and use freshly prepared solution.
2. Cell staining (suspended cells)
2.1 Centrifuge and collect cells, wash twice with PBS for 5 minutes each time. Cell density is 1 × 10~6/mL
2.2 Add 1 mL of FM working solution and incubate at room temperature for 5-30 minutes.
2.3 At 400 g, centrifuge for 3-4 minutes, discard the supernatant.
2.4 Wash the cells twice with PBS, each time for 5 minutes.
After resuspending cells in 1 mL serum-free medium or PBS, monitor them using a fluorescence microscope or flow cytometer.
3. Cell staining (adherent cells)
3.1 Culture adherent cells on sterile coverslips.
3.2 Remove the cover glass from the culture medium and aspirate excess culture medium.
3.3 Add 100 μ L of dye working solution, gently shake to completely cover the cells, and incubate for 5-30 minutes.
3.4 Remove the dye working solution, wash 2-3 times with culture medium for 5 minutes each time, and monitor using a fluorescence microscope or flow cytometer.

In this study, researchers report a distinct microenvironment within the nuclear envelope (NE) in living cells revealed by a spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br). The dye readily translocated to the NE at physiological temperature where it exhibited enhanced fluorescence when excited at 620-650 nm in contrast to 480-520 nm excitation in the endocytic pathway and in the endoplasmic reticulum (ER). In vitro data indicated that the dye reveals an enrichment of negatively charged lipids, presumably due to local phospholipid synthesis. Dual-excitation imaging of FM4-64 in relation to lamina-associated polypeptide-1-green fluorescent protein during mitosis suggested that the disassembly of NE preserves microscale lipid complexes in the ER. Convolutions of NE in cancer or primary cells were readily visualized, and killing of tumor cells by T cells was marked by sudden loss of the long-wavelength excited fluorescence in the NE coincident with apoptosis. This report of FM4-64 as the first vital dye sensitive to the NE environment opens new ways for real-time visualization and functional studies of the NE. [2]

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Aim: Conformational analysis of fluorescent styryl dyes FM 1-43 and FM 4-64 was undertaken to clarify if distinct activity-dependent labelling of single lactotrophs vesicles and plasma membrane by two dyes is associated with their structural differences. Methods: The activity-dependent labelling of single vesicles and plasma membrane by FM 1-43 and FM 4-64 was studied using confocal microscopy. The fluorescence intensity of vesicles fused with the plasma membrane, and the plasma membrane alone was measured; the ratio of their respective peak amplitudes was calculated. The conformational analysis of FM 1-43 and FM 4-64 was further undertaken by employing the Monte Carlo approach to search the conformational space of these molecules. Results: In FM 1-43 staining of vesicles and plasma membrane, the ratio of the fluorescence peak amplitudes (vesicle vs. plasma membrane) was 2.6 times higher in comparison with FM 4-64 staining. In FM 4-64 molecule the low-energy conformations are distributed in three conformational states (consisting of 3, 4 and 2 conformers respectively) in which the proportion of the molecules residing in a given state is 62%, 28% and 9% respectively. In FM 1-43 the conformation distribution is limited to just one conformational state with three approximately equally populated conformers what can be explained by greater intrinsic rigidity of the molecule. Conclusions: The observed structural characteristics of FM 1-43 molecules may account for a higher increase in quantum yield and/or binding affinity upon incorporation of the dye into the vesicle matrix and therefore stronger fluorescence emission in comparison with FM 4-64. [3]

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FM-dyes are widely used to study endocytosis, vesicle trafficking and organelle organization in living eukaryotic cells. The increasing use of FM-dyes in plant cells has provoked much debate with regard to their suitability as endocytosis markers, which organelles they stain and the precise pathways they follow through the vesicle trafficking network. A primary aim of this article is to assess critically the current status of this debate in plant cells. For this purpose, background information on the important characteristics of the FM-dyes, and of optimal dye concentrations, conditions of dye storage, and staining and imaging protocols, are provided. Particular emphasis is placed on using the FM-dyes in double labelling experiments to identity specific organelles. In this way, staining of the Golgi with FM4-64 has been demonstrated for the first time. [4]

[1]. FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel. J Neurosci. 2001 Sep 15;21(18):7013-25.

[2]. Spectral shift of fluorescent dye FM4-64 reveals distinct microenvironment of nuclear envelope in living cells. Traffic. 2006;7(12):1607-1613.

[3]. 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.

[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.

Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.[1]

C30H45N3+2.2[BR-]

607.5064

605.198

C, 59.31; H, 7.47; Br, 26.31; N, 6.92

162112-35-8

6508728

Gray to dark gray solid powder

0.377

0

3

14

35

561

0

CCN(CC)C1=CC=C(C=C1)/C=C/C=C/C=C/C2=CC=[N+](C=C2)CCC[N+](CC)(CC)CC.[Br-].[Br-]

AFVSZGYRRUMOFH-UHFFFAOYSA-L

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

3-[4-[(1E,3E,5E)-6-[4-(diethylamino)phenyl]hexa-1,3,5-trienyl]pyridin-1-ium-1-yl]propyl-triethylazanium;dibromide

FM4 64; FM464; 162112-35-8; FM4-64; N-(3-Triethylammoniopropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl)pyridinium dibromide; 3-[4-[(1E,3E,5E)-6-[4-(Diethylamino)phenyl]hexa-1,3,5-trienyl]pyridin-1-ium-1-yl]propyl-triethylazanium;dibromide; 4-((1E,3E,5E)-6-(4-(diethylamino)phenyl)hexa-1,3,5-trien-1-yl)-1-(3-(triethylammonio)propyl)pyridin-1-ium bromide; 4-(6-(4-(diethylamino)phenyl)hexa-1,3,5-trien-1-yl)-1-(3-(triethylammonio)propyl)pyridin-1-ium bromide; FM4-64

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~82.30 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (1.65 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (1.65 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)