Fluo-3, AM *CAS 121714-22-5*
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Dissociation constant (Kd, nM) | 390 |
Molecular weight | 1129.85 |
Solvent | DMSO |
Extinction coefficient (cm -1 M -1) | 86,0001 |
Excitation (nm) | 506 |
Emission (nm) | 515 |
Quantum yield | 0.151 |
Certificate of Origin | Download PDF |
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 |
Cal-520®, AM |
Fluo-8®, AM |
Calbryte™ 520 AM |
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5* |
Fluo-3, AM *Bulk package* *CAS 121714-22-5* |
Overview | ![]() ![]() |
CAS 121714-22-5 | Molecular weight 1129.85 | Dissociation constant (Kd, nM) 390 | Extinction coefficient (cm -1 M -1) 86,0001 | Excitation (nm) 506 | Emission (nm) 515 | Quantum yield 0.151 |
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
Prepare a 2 to 5 mM stock solution of Fluo-3 AM in high-quality, anhydrous DMSO.
PREPARATION OF 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 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.
- Prepare cells in growth medium overnight.
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.
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
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
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum

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
![<strong>Effect of increased [Ca<sup>2+</sup>]i on the subcellular localization of CacyBP/SIP in colon cancer SW480 cells. </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 μmol/L) for 30 min at 37°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 μm. (B) The intensity of cytosolic free intracellular Ca<sup>2+</sup> fluorescence in SW480 cells treated with ionomycin (0, 1, 2, 5, 10 μmol/L). The Fluo-3 fluorescence intensity in SW480 cells reached a plateau at 5 μmol/L and 10 μmol/L of ionomycin. SW480 cells were loaded with 20 μmol/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 μmol/L of ionomycin (***P<0.001). Source: <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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Ffluo-3-am-cas-121714-22-5%2Ffigure-for-fluo-3-am-cas-121714-22-5_cz3j2.jpg&w=3840&q=75)

Citations
Authors: Lin, Chunlong and Li, Caixia and Zhao, Jianping and Ni, Wang and Yi, Jizu
Journal: Pakistan Journal of Pharmaceutical Sciences (2020)
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
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
Authors: Xiaoyu, DONG
Journal: Plasma Science and Technology (2018): 044001
Authors: Peng, Xudong and Zhao, Guiqiu and Lin, Jing and Qu, Jianqiu and Zhang, Yingxue and Li, Cui
Journal: BMC ophthalmology (2018): 170
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
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: 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: 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: 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
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Application notes
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
A Meta-Analysis of Common Calcium Indicators
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets