logo
AAT Bioquest

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

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.

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
  1. 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
  1. 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.

  2. Prepare a 2 to 20 µM Fluo-3 AM working solution 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 solutions, 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 to 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
/=x=

Spectrum

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*50651586,00010.151
Fluo-8H™, AM495516234300.161
Fluo-8L™, AM495516234300.161
Fluo-8FF™, AM495516234300.161
Fluo-5F, AM *Cell permeant*494516--
Fluo-5N, AM *Cell permeant*494516--

Citations

View all 20 citations: Citation Explorer
Comprehensive review of indicators and techniques for optical mapping of intracellular calcium ions
Authors: Sheng, Chu-Qiao and Wu, Shuang-Shuang and Cheng, Yong-Kang and Wu, Yao and Li, Yu-Mei
Journal: Cerebral Cortex (2024): bhae346
Microfluidic organ chip of fluid--solid dynamic curved interface
Authors: Su, Haoran and Ma, Tianxiang and Liu, Xiao and Wang, Li and Shu, Fangjun and Liang, Zhuqing and Zhang, Dongrui and Zhang, Xing and Li, Kexin and Wang, Min and others,
Journal: Applied Physics Reviews (2024)
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

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
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
Page updated on November 11, 2024

Ordering information

Price
Unit size
Catalog Number21010
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Dissociation constant (Kd, 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

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

CAS

121714-22-5

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
<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.
Gallery Image 3