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Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*

Chemical structure for Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*
Chemical structure for Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*
Ordering information
Price ()
Catalog Number20550
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)345
Molecular weight1096.95
SolventDMSO
Spectral properties
Extinction coefficient (cm -1 M -1)82000
Excitation (nm)495
Emission (nm)528
Quantum yield0.161
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

OverviewpdfSDSpdfProtocol


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

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-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.
  1. Prepare cells in growth medium overnight.
  2. 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.
  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-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 mg0.5 mg1 mg5 mg10 mg
1 mM91.162 µL455.809 µL911.619 µL4.558 mL9.116 mL
5 mM18.232 µL91.162 µL182.324 µL911.619 µL1.823 mL
10 mM9.116 µL45.581 µL91.162 µL455.809 µL911.619 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

Extinction coefficient (cm -1 M -1)82000
Excitation (nm)495
Emission (nm)528
Quantum yield0.161

Citations


View all 17 citations: Citation Explorer
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
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
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
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
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
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
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)
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
HuR regulates phospholamban expression in isoproterenol-induced cardiac remodelling
Authors: Hu, Han and Jiang, Mingyang and Cao, Yangpo and Zhang, Zhuojun and Jiang, Bin and Tian, Feng and Feng, Juan and Dou, Yali and Gorospe, Myriam and Zheng, Ming and others,
Journal: Cardiovascular research (2020): 944--955
Influence of PEDOT: PSS Coating Thickness on the Performance of Stimulation Electrodes
Authors: Dijk, Gerwin and Ruigrok, Hermanus J and O'Connor, Rodney P
Journal: Advanced Materials Interfaces (2020): 2000675