Fluo-3, AM UltraPure grade CAS 121714-22-5

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Physical properties

Dissociation constant (K_d, nM)	390
Molecular weight	1129.85
Solvent	DMSO

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	86,000¹
Excitation (nm)	506
Emission (nm)	515
Quantum yield	0.15¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Direct upgrades

Cal-520®, AM

Fluo-8®, AM

Calbryte™ 520 AM

Alternative formats

Fluo-3, AM *CAS 121714-22-5*

Fluo-3, AM *Bulk package* *CAS 121714-22-5*

Overview SDS Protocol

Upgrade: Cal-520®, AM, Fluo-8®, AM, Calbryte™ 520 AM

CAS

121714-22-5

Molecular weight

1129.85

Dissociation constant (K_d, nM)

390

Extinction coefficient (cm ^-1 M ^-1)

86,000¹

Excitation (nm)

506

Emission (nm)

515

Quantum yield

0.15¹

Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Fluo-3 and Rhod-2 are most commonly used among the visible light-excitable calcium indicators. Fluo-3 indicators are widely used in flow cytometry and confocal laser-scanning microscopy. More recently, Fluo-3, AM has been extensively used in cell-based high-throughput screening assays for functional GPCR assays. Fluo-3 is essentially nonfluorescent unless bound to Ca2+ and exhibits a quantum yield at saturating Ca2+ of ~0.14 and a Kd for Ca2+ of 390 nM.

Platform

Fluorescence microscope

Excitation	FITC
Emission	FITC
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	490
Emission	525
Cutoff	515
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode/Programmable liquid handling

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Fluo-3 AM *UltraPure grade* Stock Solution

Prepare a 2 to 5 mM stock solution of Fluo-3 AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Fluo-3 AM *UltraPure grade* Working Solution

On the day of the experiment, either dissolve Fluo-3 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Fluo-3 AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Fluo-3 AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
Note If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ probenecid products, including water-soluble, sodium salt, and stabilized solution, can be purchased from AAT Bioquest.

SAMPLE EXPERIMENTAL PROTOCOL

Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.

Prepare cells in growth medium overnight.
On the next day, add 1X Fluo-3 AM working solution into your cell plate.
Note If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
Note Incubating the dye for longer than 2 hours can improve signal intensities in certain cell lines.
Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at 490/525 nm cutoff 515 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fluo-3, AM *UltraPure grade* *CAS 121714-22-5* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	88.507 µL	442.537 µL	885.073 µL	4.425 mL	8.851 mL
5 mM	17.701 µL	88.507 µL	177.015 µL	885.073 µL	1.77 mL
10 mM	8.851 µL	44.254 µL	88.507 µL	442.537 µL	885.073 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	86,000¹
Excitation (nm)	506
Emission (nm)	515
Quantum yield	0.15¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield
Fluo-4 AM Ultrapure Grade CAS 273221-67-3	495	528	82000	0.16¹
Fluo-3FF, AM UltraPure grade Cell permeant	506	515	86,000¹	0.15¹
Fluo-8H™, AM	495	516	23430	0.16¹
Fluo-8L™, AM	495	516	23430	0.16¹
Fluo-8FF™, AM	495	516	23430	0.16¹
Fluo-5F, AM Cell permeant	494	516	-	-
Fluo-5N, AM Cell permeant	494	516	-	-

Images

Figure 1. Chemical structure for Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*

Citations

View all 16 citations: Citation Explorer

A Dipeptidyl Peptidase IV Inhibitory Peptide Relieves Palmitic Acid-Induced Endoplasmic Reticulum Stress in HepG2 Cells Independently of Inhibiting Dipeptidyl Peptidase IV Activity
Authors: Jin, Ritian and Ren, Haowei and Liao, Minhe and Shang, Jiaqi and Wang, Dangfeng and Li, Meng and Liu, Ning
Journal: Food \& Function (2021)

An Effective Inhibitory Strategy of Low Steady Magnetic Field on Ovarian Cancer
Authors: Li, Xiaodi and Fang, Yanwen and Fang, Zhicai and Wang, Ping and Zhu, Jun
Journal: (2021)

CaMK4-dependent phosphorylation of Akt/mTOR underlies Th17 excessive activation in experimental autoimmune prostatitis
Authors: Zhan, Chang-Sheng and Chen, Jia and Chen, Jing and Zhang, Li-Gang and Liu, Yi and Du, He-Xi and Wang, Hui and Zheng, Mei-Juan and Yu, Zi-Qiang and Chen, Xian-Guo and others,
Journal: The FASEB Journal (2020): 14006--14023

Phospholipase Cγ2 is critical for Ca 2+ flux and cytokine production in anti-fungal innate immunity of human corneal epithelial cells
Authors: Peng, Xudong and Zhao, Guiqiu and Lin, Jing and Qu, Jianqiu and Zhang, Yingxue and Li, Cui
Journal: BMC ophthalmology (2018): 170

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
Authors: Wu, Yanjiao and Xu, Xiaoli and Ma, Lunkun and Yi, Qian and Sun, Weichao and Tang, Liling
Journal: The International Journal of Biochemistry & Cell Biology (2017)

Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells
Authors: Lu, Jiang and Yao, Xue-qin and Luo, Xin and Wang, Yu and Chung, Sookja Kim and Tang, He-xin and Cheung, Chi Wai and Wang, Xian-yu and Meng, Chen and Li, Qing and others, undefined
Journal: Neural Regeneration Research (2017): 945

Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels
Authors: Yang, Gang and Xiao, Zhenghua and Ren, Xiaomei and Long, Haiyan and Ma, Kunlong and Qian, Hong and Guo, Yingqiang
Journal: Scientific Reports (2017): 41781

Dexmedetomidine reduces hypoxia/reoxygenation injury by regulating mitochondrial fission in rat hippocampal neurons
Authors: Liu, Jia and Du, Qing and Zhu, He and Li, Yu and Liu, Maodong and Yu, Shoushui and Wang, Shilei
Journal: Int J Clin Exp Med (2017): 6861--6868

Calreticulin regulates TGF-$\beta$1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
Authors: Wu, Yanjiao and Xu, Xiaoli and Ma, Lunkun and Yi, Qian and Sun, Weichao and Tang, Liling
Journal: The International Journal of Biochemistry \& Cell Biology (2017): 103--113

Di (2-ethylhexyl) phthalate-induced apoptosis in rat INS-1 cells is dependent on activation of endoplasmic reticulum stress and suppression of antioxidant protection
Authors: Sun, Xia and Lin, Yi and Huang, Qiansheng and Shi, Junpeng and Qiu, Ling and Kang, Mei and Chen, Yajie and Fang, Chao and Ye, Ting and Dong, Sijun
Journal: Journal of cellular and molecular medicine (2015): 581--594

References

View all 53 references: Citation Explorer

A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220

Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy
Authors: Orlicky J, Sulova Z, Dovinova I, Fiala R, Zahradnikova A, Jr., Breier A.
Journal: Gen Physiol Biophys (2004): 357

Comparison of human recombinant adenosine A2B receptor function assessed by Fluo-3-AM fluorometry and microphysiometry
Authors: Patel H, Porter RH, Palmer AM, Croucher MJ.
Journal: Br J Pharmacol (2003): 671

Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics
Authors: Loughrey CM, MacEachern KE, Cooper J, Smith GL.
Journal: Cell Calcium (2003): 1

A sensitive method for the detection of foot and mouth disease virus by in situ hybridisation using biotin-labelled oligodeoxynucleotides and tyramide signal amplification
Authors: Zhang Z, Kitching P.
Journal: J Virol Methods (2000): 187

Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) chang
Authors: Rockwell PL, Storey BT.
Journal: Mol Reprod Dev (2000): 335

MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells
Authors: Cantz T, Nies AT, Brom M, Hofmann AF, Keppler D.
Journal: Am J Physiol Gastrointest Liver Physiol (2000): G522

Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca(2+)concentrations distribution in living plant tissue
Authors: Walczysko P, Wagner E, Albrechtova JT.
Journal: Cell Calcium (2000): 23

[Ca2+]i following extrasystoles in guinea-pig trabeculae microinjected with fluo-3 - a comparison with frog skeletal muscle fibres
Authors: Wohlfart B., undefined
Journal: Acta Physiol Scand (2000): 1

Determination of the intracellular dissociation constant, K(D), of the fluo-3. Ca(2+) complex in mouse sperm for use in estimating intracellular Ca(2+) concentrations
Authors: Rockwell PL, Storey BT.
Journal: Mol Reprod Dev (1999): 418