Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*
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Catalog Number | |
Availability | In stock |
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Bulk request | Inquire |
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Dissociation constant (Kd, nM) | 345 |
Molecular weight | 1096.95 |
Solvent | DMSO |
Extinction coefficient (cm -1 M -1) | 82000 |
Excitation (nm) | 495 |
Emission (nm) | 528 |
Quantum yield | 0.161 |
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 |
Overview | ![]() ![]() |
CAS 273221-67-3 | Molecular weight 1096.95 | Dissociation constant (Kd, nM) 345 | Extinction coefficient (cm -1 M -1) 82000 | Excitation (nm) 495 | Emission (nm) 528 | Quantum yield 0.161 |
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-4 AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Fluo-4 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Fluo-4 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-4 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-4 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-4 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 | 91.162 µL | 455.809 µL | 911.619 µL | 4.558 mL | 9.116 mL |
5 mM | 18.232 µL | 91.162 µL | 182.324 µL | 911.619 µL | 1.823 mL |
10 mM | 9.116 µL | 45.581 µL | 91.162 µL | 455.809 µL | 911.619 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) | 82000 |
Excitation (nm) | 495 |
Emission (nm) | 528 |
Quantum yield | 0.161 |
Product Family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield |
Fluo-3, AM *CAS 121714-22-5* | 506 | 515 | 86,0001 | 0.151 |
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5* | 506 | 515 | 86,0001 | 0.151 |
Fluo-3, AM *Bulk package* *CAS 121714-22-5* | 506 | 515 | 86,0001 | 0.151 |
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 | - | - |
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Citations
Authors: Goretzko, Jonas and Pauels, Inga and Heitzig, Nicole and Thomas, Katharina and Kardell, Marina and Na{\ss}, Johannes and Krogsaeter, Einar Kleinhans and Schloer, Sebastian and Spix, Barbara and Matos, Anna L{\'\i}via Linard and others,
Journal: Cell Reports (2023): 113501
Authors: Shiba, Naoko and Yang, Xiao and Sato, Mitsuto and Kadota, Shin and Suzuki, Yota and Agata, Masahiro and Nagamine, Kohei and Izumi, Masaki and Honda, Yusuke and Koganehira, Tomoya and others,
Journal: Molecular Therapy-Nucleic Acids (2023)
Authors: Hu, Jin and Chen, Qiang and Zhu, Hongrui and Hou, Lichao and Liu, Wei and Yang, Qihua and Shen, Huidan and Chai, Guolin and Zhang, Boxin and Chen, Shaoxuan and others,
Journal: Neuron (2023): 1--15
Authors: Dong, Shuangshan and Kifune, Takashi and Kato, Hiroki and Wang, Lu and Kong, Jun and Hirofuji, Yuta and Sun, Xiao and Sato, Hiroshi and Ito, Yosuke and Kato, Takahiro A and others,
Journal: Biochemical and Biophysical Research Communications (2023)
Authors: Zhu, Ruohan and Meng, Qian and Zhang, Huijun and Zhang, Ge and Huang, Lina SM and Xu, Yan and Schooley, Robert T and An, Jing and Huang, Ziwei
Journal: European Journal of Medicinal Chemistry (2022): 114797
Authors: Visa, Anna and Alza, L{\'\i}a and Cant{\'\i}, Carles and Herreros, Judit
Journal: Biomedicine \& Pharmacotherapy (2022): 112881
Authors: Kanno, Shoichiro and Peng, Zugui and Shimba, Kenta and Miyamoto, Yoshitaka and Yagi, Tohru
Journal: IEEJ Transactions on Electronics, Information and Systems (2021): 620--626
Authors: Oguro, Ami and Sugitani, Atsushi and Kobayashi, Yukino and Sakuma, Rika and Imaoka, Susumu
Journal: The Journal of Toxicological Sciences (2021): 1--10
Authors: Dijk, Gerwin and Ruigrok, Hermanus J and O'Connor, Rodney P
Journal: Advanced Materials Interfaces (2021): 2100214
Authors: Zhao, Fang and Zou, Xiaohan and Li, Shaoheng and He, Jing and Xi, Chuchu and Tang, Qinglian and Wang, Yujing and Cao, Zhengyu
Journal: Toxins (2021): 33
Application notes
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
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Cal-520 ® , Cal-590 ™, and Cal-630™ Calcium Detection Reagents
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