Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
Quotation | Request |
International | See distributors |
Shipping | Standard overnight for United States, inquire for international |
Physical properties
Dissociation constant (Kd, nM) | 345 |
Molecular weight | 1096.95 |
Solvent | DMSO |
Spectral properties
Extinction coefficient (cm -1 M -1) | 82000 |
Excitation (nm) | 495 |
Emission (nm) | 528 |
Quantum yield | 0.161 |
Storage, safety and handling
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 |
Direct upgrades
Cal-520®, AM |
Fluo-8®, AM |
Calbryte™ 520 AM |
Related products
Overview | ![]() ![]() |
See also: Calcium Indicators, Chemical Reagents, Classic Dyes, Fluo-4, Fluoresceins, Intracellular Ions, Physiological Probes
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 |
Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Fluo-3 and Fluo-4 are most commonly used among the visible light-excitable calcium indicators. Fluo-4 is an analog of Fluo-3 with the two chlorine substituents replaced by fluorines, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. However, Fluo-3 AM and Fluo-4 AM are only moderately fluorescent in live cells upon esterase hydrolysis, and require harsh cell loading conditions to maximize their cellular calcium responses. Fluo-8® and Cal-520® calcium dyes have been developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelength of maximum excitation @ ~490 nm and maximum emission @ ~520 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-4 AM *UltraPure grade* Stock Solution
Prepare a 2 to 5 mM stock solution of Fluo-4 AM in high-quality, anhydrous DMSO.PREPARATION OF WORKING SOLUTION
Fluo-4 AM *UltraPure grade* Stock 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 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-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 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-4 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-4 AM *Ultrapure Grade* *CAS 273221-67-3* 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 | 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
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Spectrum
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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 | - | - |
Show More (1) |
Citations
View all 20 citations: Citation Explorer
Microglial Piezo1 senses Ab fibril stiffness to restrict Alzheimer’s disease
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: 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
HIV-1 gp120-CXCR4 recognition probed with synthetic nanomolar affinity D-peptides containing fragments of gp120 V3 loop
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: 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
Tetralol derivative NNC-55-0396 induces glioblastoma cell death by activating IRE1$\alpha$, JNK1 and calcium signaling
Authors: Visa, Anna and Alza, L{\'\i}a and Cant{\'\i}, Carles and Herreros, Judit
Journal: Biomedicine \& Pharmacotherapy (2022): 112881
Authors: Visa, Anna and Alza, L{\'\i}a and Cant{\'\i}, Carles and Herreros, Judit
Journal: Biomedicine \& Pharmacotherapy (2022): 112881
Functional Analysis of Liposomes Containing Single-Walled Carbon Nanotubes (SWNTs) by Fluorescence Microscopy
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: 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
Bisphenol A stabilizes Nrf2 via Ca2+ influx by direct activation of the IP3 receptor
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: Oguro, Ami and Sugitani, Atsushi and Kobayashi, Yukino and Sakuma, Rika and Imaoka, Susumu
Journal: The Journal of Toxicological Sciences (2021): 1--10
PEDOT: PSS-Coated Stimulation Electrodes Attenuate Irreversible Electrochemical Events and Reduce Cell Electropermeabilization
Authors: Dijk, Gerwin and Ruigrok, Hermanus J and O'Connor, Rodney P
Journal: Advanced Materials Interfaces (2021): 2100214
Authors: Dijk, Gerwin and Ruigrok, Hermanus J and O'Connor, Rodney P
Journal: Advanced Materials Interfaces (2021): 2100214
BmK NSPK, a Potent Potassium Channel Inhibitor from Scorpion Buthus martensii Karsch, Promotes Neurite Outgrowth via NGF-TrkA Signaling Pathway
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
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
Sinoatrial node pacemaker cells share dominant biological properties with glutamatergic neurons
Authors: Liang, Dandan and Xue, Zhigang and Xue, Jinfeng and Xie, Duanyang and Xiong, Ke and Zhou, Huixing and Zhang, Fulei and Su, Xuling and Wang, Guanghua and Zou, Qicheng and others,
Journal: Protein \& Cell (2021): 1--12
Authors: Liang, Dandan and Xue, Zhigang and Xue, Jinfeng and Xie, Duanyang and Xiong, Ke and Zhou, Huixing and Zhang, Fulei and Su, Xuling and Wang, Guanghua and Zou, Qicheng and others,
Journal: Protein \& Cell (2021): 1--12
A novel Ca2+ indicator for long-term tracking of intracellular calcium flux
Authors: Liao, Jinfang and Patel, Deven and Zhao, Qin and Peng, Ruogu and Guo, Haitao and Diwu, Zhenjun
Journal: BioTechniques (2021): 271--277
Authors: Liao, Jinfang and Patel, Deven and Zhao, Qin and Peng, Ruogu and Guo, Haitao and Diwu, Zhenjun
Journal: BioTechniques (2021): 271--277
Thioredoxin-1 regulates calcium homeostasis in MPP+/MPTP-induced Parkinson's disease models
Authors: Zhang, Xianwen and Deng, Ruhua and Zhang, Se and Deng, Juan and Jia, Jing Jing and Sun, Bo and Zhou, Xiaoshuang and Bai, Jie
Journal: European Journal of Neuroscience (2021)
Authors: Zhang, Xianwen and Deng, Ruhua and Zhang, Se and Deng, Juan and Jia, Jing Jing and Sun, Bo and Zhou, Xiaoshuang and Bai, Jie
Journal: European Journal of Neuroscience (2021)
Application notes
A Meta-Analysis of Common Calcium Indicators
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Cal-520 ® , Cal-590 ™, and Cal-630™ Calcium Detection Reagents
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Cal-520 ® , Cal-590 ™, and Cal-630™ Calcium Detection Reagents
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents