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 FM 1-43 working solution using preheated HBSS solution.
Note: Please adjust the concentration of FM 1-43 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.

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].

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

FM 1-43 dye is an organic bromide salt, a quaternary ammonium salt, a tertiary amine and a pyridinium salt. It has a role as a fluorochrome. It contains a FM 1-43(2+).

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Fluorescent Dyes: Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags

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There are a striking number of similarities between the characteristics of FM1-43 dye loading and the mechanisms of aminoglycoside accumulation or toxicity in mouse cochlear cultures. The accumulation of [3H]-gentamicin in cochlear hair cells and the morphological effects of neomycin are considerably reduced at low temperature (4°C) and absent in Myo7a mutants (Richardson et al., 1997). The morphological effects of neomycin also manifest themselves rapidly. They can be blocked by elevated extracellular calcium (Richardson and Russell, 1991) and, as shown in this study, can be prevented by pretreatment with calcium chelators. Furthermore, a gradient of sensitivity to neomycin from base to apex of the cochlea, similar to that observed for FM1-43 dye loading, is also seen in cochlear cultures (Richardson and Russell, 1991). Finally, FM1-43 is able to reduce the ototoxic effect of neomycin, indicating that the two compounds compete for the same entry mechanism. These observations, combined with the evidence presented above, suggest that FM1-43 and aminoglycoside antibiotics may both enter hair cells via the transducer channel. The latter possibility has been suggested previously (Kroese et al., 1989). The finding that FM1-43 does not block the channel from the interior of the cell at concentrations where it blocks strongly from the extracellular side reveals that it passes preferentially in one direction. If the same holds for the aminoglycoside antibiotics, it would explain why both FM1-43 and aminoglycosides accumulate in hair cells and do not unload after exposure. The known differential sensitivity of various hair-cell types to aminoglycoside antibiotics may also be explained by differences in channel open probability at rest. Basal-coil outer hair cells in the mammalian cochlea are among the most sensitive to aminoglycoside damage (Hawkins, 1976), and there is evidence that in vivo they maintain their hair bundles biased with 50%, as opposed to the more usual 5–10%, of their transducer channels open at rest (Russell and Kössl, 1992). In conclusion, the results of this study provide strong evidence that FM1-43 enters the apical pole of sensory hair cells in the mammalian cochlea via the mechanotransducer channel and suggest that the ototoxic aminoglycoside antibiotics may share the same entry pathway.[1]

C30H49N3+2.2[BR-]

611.53816

609.229

C, 58.92; H, 8.08; Br, 26.13; N, 6.87

149838-22-2

6508724

Orange to red solid powder

0.825

0

3

16

35

480

0

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

VZUVCAGXYLMFEC-UHFFFAOYSA-L

InChI=1S/C30H49N3.2BrH/c1-6-11-23-32(24-12-7-2)30-18-16-28(17-19-30)14-15-29-20-25-31(26-21-29)22-13-27-33(8-3,9-4)10-5;;/h14-21,25-26H,6-13,22-24,27H2,1-5H3;2*1H/q+2;;/p-2

3-[4-[(E)-2-[4-(dibutylamino)phenyl]ethenyl]pyridin-1-ium-1-yl]propyl-triethylazanium;dibromide

FM1-43; 149838-22-2; Fm1 43; NerveGreen C1; N-(3-(triethylammonium)propyl)-4-(4-(dibutylamino)styryl)pyridinium; UNII-83IGZ86I9D; FM 1-43; 83IGZ86I9D;

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 (~81.76 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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