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

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Physical properties

Dissociation constant (K_d, 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

Overview SDS Protocol

Upgrade: Cal-520®, AM, Fluo-8®, AM, Calbryte™ 520 AM

CAS

273221-67-3

Molecular weight

1096.95

Dissociation constant (K_d, 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

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

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	-	-
Rhod-4™, AM	523	551	-	0.1¹
Show More (1)

Images

Figure 1. Chemical structure for Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*

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

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

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

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)