Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*
| Overview |  SDSProtocol |
See also: Calcium Indicators, Chemical Reagents, Classic Dyes, Fluo-3, Fluoresceins, Intracellular Ions, Physiological Probes
CAS 121714-22-5 | Molecular weight 1129.85 | Dissociation constant (Kd, nM) 390 | Excitation (nm) 507 | Emission (nm) 516 | 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
| Excitation (nm) | 507 |
| Emission (nm) | 516 |
| 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* | 507 | 516 | - | 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-2, AM | - | - | - | - |
| 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)
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)
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
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
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)
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Authors: Rockwell PL, Storey BT.
Journal: Mol Reprod Dev (1999): 418
Application notes
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents
AssayWise
A Simple End-Point Calcium Assay Using a Green Fluorescent Indicator Fluo-8E™, AM
A Simple End-Point Calcium Assay Using a Green Fluorescent Indicator Fluo-8E™, AM
A Simple End-Point Calcium Assay Using a Green Fluorescent Indicator Fluo-8E™, AM
A Simple End-Point Calcium Assay Using a Green Fluorescent Indicator Fluo-8E™, AM
A Simple End-Point Calcium Assay Using a Green Fluorescent Indicator Fluo-8E™, AM