Vari Fluor 633-Phalloidin

Overview of the Product: One poison found in the lethal Amanita mushroom is called phalloidin. It is a bicyclic peptide that attaches itself to F-actin alone. Phalloidin that has been labeled with a fluorescent dye makes it very convenient to investigate the distribution of F-actin. An uncommon thioether bridge that forms an internal ring structure between cysteine and tryptophan is found inside phalloidin. This thioether is broken down by rising pH, which causes phalloidin to lose its attraction to actin. Variable Myofilament protein-specific labeling is possible with Fluor-Phalloidin, a fluorescent derivative of VF series dye-labeled Phalloidin. Within the cells, the fluorescence can last for over a week and is quite persistent. The amount of dye used to stain a slide filled with cells is measured in units (T) of VF-Phalloidin. Useful instructions Making the working solution for VF-Phalloidin 1.1 Making the storage solution To make the mother solution, dissolve the freeze-dried powder in the brown tube using the required volume of methanol or sterile water. 0.25 mL of dissolving solution should be added for the 50T specified dye. Add 1.5 mL of the dissolving solution for the 300T specified dye. It is advised to aliquot the VF-Phalloidin storage solution and keep it at -20°C or -80°C in the dark. It is advised to dissolve it in methanol for extended storage. 1.2 Getting the working solution ready: The stock solution can be diluted using PBS or hot serum-free cell culture media. Dilution ratio: 1:40–1:200, or 1–5 μL of VF-Phalloidin stock solution for each 200 μL of PBS. Please ensure that the VF-Phalloidin working fluid is prepared for use by adjusting its concentration to suit the specific circumstances. 2. Adherent cells stained with cell dye 2.1 On sterile coverslips, culture the adhering cells and give them three PBS washes. 2.2 Fix the cells for 20 minutes at room temperature using a PBS solution containing 4% formaldehyde. REMEMBER: During fixation, methanol might harm actin. Therefore, it's recommended to stay away from fixatives that include any amount of methanol. Methanol-free formaldehyde is a recommended fixative. 2.3 Rinse cells three times in PBS. 2.4 Use 0.4% Triton X-100 in PBS solution to permeabilize cells for 10 minutes at room temperature. 2.5 Repeatedly wash cells in PBS. 2.6 Cover the cells fully with 200 μL of the dye working solution, shake gently, and let it sit at room temperature in the dark for 15 to 45 minutes. Note: The conditions of the sample might be taken into account while adjusting the chromosomal volume. The coverslips can be kept in a covered container to prevent the dye solution from evaporating during the incubation period. 2.7 Aspirate out the dye working solution and repeat the two to three PBS or culture medium washes. 2.8 Apply a flow cytometer or fluorescence microscope to view in the appropriate channel. Observations 1. To prevent recurrent freezing and thawing, it is advised that VF-Phalloidin storage solution be kept in the dark at -20°C or -80°C after aliquots. It keeps for a month at -20°C and six months at -80°C. 2. Live cell labeling is not a common application for fluorescently tagged phalloidin since it is not cell permeable. Nonetheless, several investigations suggest that pinocytosis or other unidentified processes could be used to mark live cells. When staining live cells, additional dye is usually needed. An alternative method for tracking actin distribution and cell mobility is to inject fluorescently labeled phalloidin into the cells. 3. Before beginning any further tests, kindly centrifuge the result until it reaches the bottom of the tube. 4. This product is exclusively intended for professional use in scientific research; it cannot be used in food or medication, nor can it be utilized for clinical diagnosis or treatment. 5. Please wear disposable gloves and a lab coat for your health and safety.

[1]. Borovikov YuS, et al. The effect of phallotoxins on the structure of F-actin in myosin-free ghost muscle fibres of rabbit. FEBS Lett. 1984 Oct 29;176(2):441-3.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

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.)