Fluo-3AM

Cat No.:V33557 Purity: ≥98%

COA Product Data Instructions for use SDS

Fluo-3 AM is a fluorescent chelator of Ca2+ with high affinity for calcium.

Fluo-3AM Chemical Structure CAS No.: 121714-22-5
Product category: New2

This product is for research use only, not for human use. We do not sell to patients.

Size	Price	Stock	Qty
1mg
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Citations by Top Journals: Nature, Cell, Science, etc.

Top Publications Citing lnvivochem Products

Nature 2021, 597(7874):119-125.

Cell 2020,183:1202-1218.e25.

Science Adv 2022: 8, eabm9427.

Nature 597, 119–125 (2021)

Cell Stem Cell 2020,26(6):845-861.e12.

Science Trans Med 2023,15(701):eadd7872.

STTT 2023,8(1):91.

Cell Reports 2023,42(4):112313

Science Trans Med 2023, 15: eadd7872.

Cancer Cell 2021,39(4):529-547.e7.

Cancer Discov 2022,12(4):1106-1127.

Immunity 2023,56(3):620-634.e11.

J Am Chem Soc 2022,144:21831-21836.

Cell 2020,183:1202-1218.e25.

Science Adv 2022:8,eabm9427.

Nature 2021,597(7874):119-125.

Cell 2020,183:1202-1218.e25.

PNAS Nexus 2022,1(3):pgac063.

ACS Nano 2023,17(10):9110-9125.

Biomaterial 2023 Sep16:302:122333.

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Related Products

Product Description

Fluo-3 AM is a fluorescent chelator of Ca2+ with high affinity for calcium. Fluo-3 AM can specifically recognize intracellular calcium ions with high sensitivity and low toxicity to cells. By increasing AM acetyl methyl ester, it can enter the cells well and be retained after being cleaved by intracellular esterases. It combines with calcium ions in cells to produce strong fluorescence.

Biological Activity I Assay Protocols (From Reference)

ln Vitro	1. Preparation of Fluo-3 AM working solution 1.1 Use anhydrous DMSO to prepare a stock solution, and use Fluo-3 AM to prepare a 10 mM stock solution. Note: It is recommended to store the Fluo-3 AM storage solution at -20°C or -80 after dispensing. 1.2 Characteristics of the working solution Use the pre-made HBSS storage solution to prepare a 1-10 μM Fluo-3 AM working solution. Note: Please adjust the concentration of Fluo-3 AM working solution according to the real condition, and 2. Suspended cell staining (2.1): Centrifuge cells, add PBS, then wash twice for five minutes each time. The density is 1×106/mL 2.2 Add 1 mL Fluo-3 AM working solution, use the freshly prepared solution first. 5-30 minutes. 2.3 Centrifuge at 400g for 3-4 minutes and discard the supernatant. 2.4 Wash the cells twice with PBS, giving them five minutes each time. 2.5 After resuspending the cells in 1 mL HBSS, utilize a fluorescence microscope or flow cytometry 3. Cell staining (adherent cells) 3.1 Culture the adherent cells on sterile coverslips. 3.2 Remove the coverslip from the cells and aspirate a tiny amount of cells. 3.3 Add 100 μL of dye working solution and wave your hand to completely cover the cells for 5-30 minutes. 3.4 Aspirate the dye working solution, wash with culture media 2-3 times, 5 minutes each time, and view with a fluorescence microscope.
References	[1]. Loughrey, C. M., MacEachern, K. E., Cooper, J., & Smith, G. L. (2003). Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics. Cell Calcium, 34(1), 1–9. doi:10.1016/s0143-4160(03)00012-5.

ln Vitro

1. Preparation of Fluo-3 AM working solution 1.1 Use anhydrous DMSO to prepare a stock solution, and use Fluo-3 AM to prepare a 10 mM stock solution. Note: It is recommended to store the Fluo-3 AM storage solution at -20°C or -80 after dispensing. 1.2 Characteristics of the working solution Use the pre-made HBSS storage solution to prepare a 1-10 μM Fluo-3 AM working solution. Note: Please adjust the concentration of Fluo-3 AM working solution according to the real condition, and 2. Suspended cell staining (2.1): Centrifuge cells, add PBS, then wash twice for five minutes each time. The density is 1×106/mL 2.2 Add 1 mL Fluo-3 AM working solution, use the freshly prepared solution first. 5-30 minutes. 2.3 Centrifuge at 400g for 3-4 minutes and discard the supernatant. 2.4 Wash the cells twice with PBS, giving them five minutes each time. 2.5 After resuspending the cells in 1 mL HBSS, utilize a fluorescence microscope or flow cytometry 3. Cell staining (adherent cells) 3.1 Culture the adherent cells on sterile coverslips. 3.2 Remove the coverslip from the cells and aspirate a tiny amount of cells. 3.3 Add 100 μL of dye working solution and wave your hand to completely cover the cells for 5-30 minutes. 3.4 Aspirate the dye working solution, wash with culture media 2-3 times, 5 minutes each time, and view with a fluorescence microscope.

References

[1]. Loughrey, C. M., MacEachern, K. E., Cooper, J., & Smith, G. L. (2003). Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics. Cell Calcium, 34(1), 1–9. doi:10.1016/s0143-4160(03)00012-5.

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula	C51H50CL2N2O23
Molecular Weight	1129.85
Exact Mass	1128.218
CAS #	121714-22-5
PubChem CID	5086914
Appearance	Brown to reddish brown solid powder
Density	1.5±0.1 g/cm3
Boiling Point	1075.8±65.0 °C at 760 mmHg
Flash Point	604.4±34.3 °C
Vapour Pressure	0.0±0.3 mmHg at 25°C
Index of Refraction	1.631
LogP	5.4
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	25
Rotatable Bond Count	36
Heavy Atom Count	78
Complexity	2220
Defined Atom Stereocenter Count	0
InChi Key	PTPUOMXKXCCSEN-UHFFFAOYSA-N
InChi Code	InChI=1S/C51H50Cl2N2O23/c1-28-7-9-39(54(19-47(62)74-24-69-30(3)57)20-48(63)75-25-70-31(4)58)45(13-28)66-11-12-67-46-14-34(8-10-40(46)55(21-49(64)76-26-71-32(5)59)22-50(65)77-27-72-33(6)60)51-35-15-37(52)41(61)17-42(35)78-43-18-44(38(53)16-36(43)51)73-23-68-29(2)56/h7-10,13-18H,11-12,19-27H2,1-6H3
Chemical Name	acetyloxymethyl 2-[2-[2-[5-[3-(acetyloxymethoxy)-2,7-dichloro-6-oxoxanthen-9-yl]-2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]-N-[2-(acetyloxymethoxy)-2-oxoethyl]-4-methylanilino]acetate
HS Tariff Code	2934.99.9001
Storage	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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility Data

Solubility (In Vitro)	DMSO : ~12.5 mg/mL (~11.06 mM)
Solubility (In Vivo)	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. Injection Formulations (e.g. IP/IV/IM/SC) 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). View More 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) Oral Formulations 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). View More 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 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. (Please use freshly prepared in vivo formulations for optimal results.)

Solubility (In Vitro)

DMSO : ~12.5 mg/mL (~11.06 mM)

Solubility (In Vivo)

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.

Injection Formulations
(e.g. IP/IV/IM/SC)

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).

View More

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)

Oral Formulations

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).

View More

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

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.

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	0.8851 mL	4.4254 mL	8.8507 mL
	5 mM	0.1770 mL	0.8851 mL	1.7701 mL
	10 mM	0.0885 mL	0.4425 mL	0.8851 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

Calculator

Mass

Concentration

Volume

Molecular Weight*

Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

Calculate the Mass of a compound required to prepare a solution of known volume and concentration
Calculate the Volume of solution required to dissolve a compound of known mass to a desired concentration
Calculate the Concentration of a solution resulting from a known mass of compound in a specific volume

See Example

An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?

Enter 350.26 in the Molecular Weight (MW) box
Enter 10 in the Concentration box and choose the correct unit (mM)
Enter 5 in the Volume box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

Concentration (Start)

Volume (Start)

Concentration (End)

Volume (End)

Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:

Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
Enter 25 into the Concentration (End) box and select the correct unit (mM)
Enter 25 into the Volume (End) box and choose the correct unit (mL)
Click the “Calculate” button
The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

Molecular formula

Total molecular weight

g/mol

Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4 c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter the chemical/molecular formula and click the “Calculate’ button.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

Volume (to add to vial)

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Desired Reconstitution Concentration

Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

Enter the mass of the reagent and the desired reconstitution concentration as well as the correct units
Click the “Calculate” button
The answer appears in the Volume (to add to vial) box

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

Dosage mg/kg

Average weight of animals g

Dosing volume per animal μL

Number of animals

Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)

% DMSO

% Tween 80

% ddH₂O

Calculation results

Working concentration： mg/mL；

Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O，mix and clarify.

Note： (1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.