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

<strong>Effect of increased [Ca<sup>2+</sup>]i on the subcellular localization of CacyBP/SIP in colon cancer SW480 cells.&nbsp;</strong>(A) Effect of different concentrations of ionomycin on the localization of endogenous CacyBP/SIP. Cells were treated with ionomycin for 30 min, followed by immunostaining using anti-CacyBP/SIP, and were imaged with confocal microscopy. CacyBP/SIP was translocated to the perinuclear region in SW480 cells. After stimulation with an increasing amount of ionomycin (0, 1, 2, 5, 10 &mu;mol/L) for 30 min at 37&deg;C, SW480 cells were fixed and immunostained using CacyBP/SIP MAb (panels a, d, g, j, and m), and nuclei were labelled with DAPI (panels b, e, h, k, and n). The merged images are shown in panels c, f, i, l, and o. The scale bar represents 50 &mu;m. (B) The intensity of cytosolic free intracellular Ca<sup>2+</sup> fluorescence in SW480 cells treated with ionomycin (0, 1, 2, 5, 10 &mu;mol/L). The Fluo-3 fluorescence intensity in SW480 cells reached a plateau at 5 &mu;mol/L and 10 &mu;mol/L of ionomycin. SW480 cells were loaded with 20 &mu;mol/L of Fluo-3/AM for 45 min under a confocal microscope (495 nm). The fluorescence was captured every 2 sec and recorded for 3 min. (C) The bar chart shows the intracellular Fluo-3 intensity. Ca<sup>2+</sup> concentration is increased by treatment with 2, 5, and 10 &mu;mol/L of ionomycin (***P&lt;0.001). Source:&nbsp;<strong>The effect of S100A6 on nuclear translocation of CacyBP/SIP in colon cancer cells</strong> by Shanshan Feng et al., <em>PLOS</em>, March 2018.
<strong>Effect of increased [Ca<sup>2+</sup>]i on the subcellular localization of CacyBP/SIP in colon cancer SW480 cells.&nbsp;</strong>(A) Effect of different concentrations of ionomycin on the localization of endogenous CacyBP/SIP. Cells were treated with ionomycin for 30 min, followed by immunostaining using anti-CacyBP/SIP, and were imaged with confocal microscopy. CacyBP/SIP was translocated to the perinuclear region in SW480 cells. After stimulation with an increasing amount of ionomycin (0, 1, 2, 5, 10 &mu;mol/L) for 30 min at 37&deg;C, SW480 cells were fixed and immunostained using CacyBP/SIP MAb (panels a, d, g, j, and m), and nuclei were labelled with DAPI (panels b, e, h, k, and n). The merged images are shown in panels c, f, i, l, and o. The scale bar represents 50 &mu;m. (B) The intensity of cytosolic free intracellular Ca<sup>2+</sup> fluorescence in SW480 cells treated with ionomycin (0, 1, 2, 5, 10 &mu;mol/L). The Fluo-3 fluorescence intensity in SW480 cells reached a plateau at 5 &mu;mol/L and 10 &mu;mol/L of ionomycin. SW480 cells were loaded with 20 &mu;mol/L of Fluo-3/AM for 45 min under a confocal microscope (495 nm). The fluorescence was captured every 2 sec and recorded for 3 min. (C) The bar chart shows the intracellular Fluo-3 intensity. Ca<sup>2+</sup> concentration is increased by treatment with 2, 5, and 10 &mu;mol/L of ionomycin (***P&lt;0.001). Source:&nbsp;<strong>The effect of S100A6 on nuclear translocation of CacyBP/SIP in colon cancer cells</strong> by Shanshan Feng et al., <em>PLOS</em>, March 2018.
<strong>Effect of increased [Ca<sup>2+</sup>]i on the subcellular localization of CacyBP/SIP in colon cancer SW480 cells.&nbsp;</strong>(A) Effect of different concentrations of ionomycin on the localization of endogenous CacyBP/SIP. Cells were treated with ionomycin for 30 min, followed by immunostaining using anti-CacyBP/SIP, and were imaged with confocal microscopy. CacyBP/SIP was translocated to the perinuclear region in SW480 cells. After stimulation with an increasing amount of ionomycin (0, 1, 2, 5, 10 &mu;mol/L) for 30 min at 37&deg;C, SW480 cells were fixed and immunostained using CacyBP/SIP MAb (panels a, d, g, j, and m), and nuclei were labelled with DAPI (panels b, e, h, k, and n). The merged images are shown in panels c, f, i, l, and o. The scale bar represents 50 &mu;m. (B) The intensity of cytosolic free intracellular Ca<sup>2+</sup> fluorescence in SW480 cells treated with ionomycin (0, 1, 2, 5, 10 &mu;mol/L). The Fluo-3 fluorescence intensity in SW480 cells reached a plateau at 5 &mu;mol/L and 10 &mu;mol/L of ionomycin. SW480 cells were loaded with 20 &mu;mol/L of Fluo-3/AM for 45 min under a confocal microscope (495 nm). The fluorescence was captured every 2 sec and recorded for 3 min. (C) The bar chart shows the intracellular Fluo-3 intensity. Ca<sup>2+</sup> concentration is increased by treatment with 2, 5, and 10 &mu;mol/L of ionomycin (***P&lt;0.001). Source:&nbsp;<strong>The effect of S100A6 on nuclear translocation of CacyBP/SIP in colon cancer cells</strong> by Shanshan Feng et al., <em>PLOS</em>, March 2018.
Spontaneous calcium transients analysis. (a) Ca<sup>2+</sup> fluorescence image from SVF-CMs loaded with the Ca<sup>2+</sup> indicator fluo-3/AM. (b) A calcium transient wave of the cell denoted by a white arrow in the panel &ldquo;a&rdquo; was produced through a customized MATLAB program. Scale bar&thinsp;=&thinsp;50&thinsp;&mu;m. Source: <strong>Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels </strong>by Yang et al., <em>Scientific Report,</em> Feb. 2017.
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Telephone1-800-990-8053
Fax1-800-609-2943
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Physical properties
Dissociation constant (Kd, nM)390
Molecular weight1129.85
SolventDMSO
Spectral properties
Excitation (nm)507
Emission (nm)516
Quantum yield0.151
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


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

ExcitationFITC
EmissionFITC
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation490
Emission525
Cutoff515
Recommended plateBlack 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 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 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.
  1. Prepare cells in growth medium overnight.
  2. 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.
  3. 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.
  4. 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.
  5. 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 *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 mg0.5 mg1 mg5 mg10 mg
1 mM88.507 µL442.537 µL885.073 µL4.425 mL8.851 mL
5 mM17.701 µL88.507 µL177.015 µL885.073 µL1.77 mL
10 mM8.851 µL44.254 µL88.507 µL442.537 µL885.073 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)507
Emission (nm)516
Quantum yield0.151

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*495528820000.161
Fluo-3FF, AM *UltraPure grade* *Cell permeant*507516-0.151
Fluo-8H™, AM495516234300.161
Fluo-8L™, AM495516234300.161
Fluo-8FF™, AM495516234300.161
Fluo-5F, AM *Cell permeant*494516--
Fluo-5N, AM *Cell permeant*494516--

Images


Citations


View all 18 citations: Citation Explorer
3, 4-dihydroxyacetophenone inhibits hypoxia-associated human pulmonary artery smooth muscle cell proliferation by reducing Ca 2+ influx.
Authors: Lin, Chunlong and Li, Caixia and Zhao, Jianping and Ni, Wang and Yi, Jizu
Journal: Pakistan Journal of Pharmaceutical Sciences (2020)
LncRNA ZNF503-AS1 acts as a tumor suppressor in bladder cancer by up-regulating Ca 2+ concentration via transcription factor GATA6
Authors: He, Haiqing and Wu, Shuiqing and Ai, Kai and Xu, Ran and Zhong, Zhaohui and Wang, Yinhuai and Zhang, Lei and Zhao, Xiaokun and Zhu, Xuan
Journal: Cellular Oncology (2020): 1--15
The selective estrogen receptor modulator raloxifene mitigates the effect of all-trans-retinal toxicity in photoreceptor degeneration
Authors: Getter, Tamar and Suh, Susie and Hoang, Thanh and Handa, James T and Dong, Zhiqian and Ma, Xiuli and Chen, Yuanyuan and Blackshaw, Seth and Palczewski, Krzysztof
Journal: Journal of Biological Chemistry (2019): 9461--9475
Measurement of cytoplasmic Ca2+ concentration in Saccharomyces cerevisiae induced by air cold plasma
Authors: Xiaoyu, DONG
Journal: Plasma Science and Technology (2018): 044001
Phospholipase C&gamma;2 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
The effect of S100A6 on nuclear translocation of CacyBP/SIP in colon cancer cells
Authors: Feng, Shanshan and Zhou, Qiaozhi and Yang, Bo and Li, Qianqian and Liu, Aiqin and Zhao, Yingying and Qiu, Changqing and Ge, Jun and Zhai, Huihong
Journal: PloS one (2018): e0192208
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 &amp; 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

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