Fluo-4 AM | AAT Bioquest

Fluo-4 AM

Ultrapure Grade; CAS 273221-67-3

Fluo-4 AM (ultrapure grade) is a high-purity cell-permeable calcium indicator with green fluorescence that provides exceptional signal-to-noise ratios for detecting calcium transients in live cells via microscopy and flow cytometry.

High-Affinity Calcium Indicator: K_d = 345 nM with >100-fold fluorescence increase upon Ca²⁺ binding
Single-Wavelength Detection: Ex/Em = 495/528 nm compatible with standard FITC optics
Improved Cell Loading: Enhanced AM ester formulation for efficient cytoplasmic delivery
BAPTA-Based Chelator: Fast calcium binding kinetics for real-time transient detection

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

Protocol

COA

Catalog	Size	Price
20550	1 mg	Price
20551	10x50 ug	Price
20552	5x50 ug	Price

Overview

Background

Fluo-4 AM is a cell permeable, green fluorescent calcium probe used in fluorescence microscopy and flow cytometry. It belongs to a class of intensiometric biosensor that detect calcium ions. It is a structural analog of Fluo-3, an earlier Ca²⁺ probe, wherein chlorine has been substituted with fluorine. The change results in an approximately 12 nm blue shift of the absorption spectrum, leading to an excitation peak of 495 nm. This is important as it enables Fluo-4 to more efficiently be excited by the 488 nm argon laser, yielding brighter fluorescence signals. As a consequence of this, lower quantities of the dye are required for assays, thereby resulting in decreased experimental cytotoxicity.

Usage

Fluo-4 AM has a molecular weight of 1096.95 Daltons. It has a calcium binding affinity (K_d Ca²⁺) of 345 nM. A stock solution can be prepared by dissolving Fluo-4 AM in DMSO (2 to 5 mM). To load into cells, prepare a working solution of 2 to 20 µM by diluting the stock solution with a buffer, such as HHBS with 0.04% Pluronic® F-127.

Excitation / Emission

Fluo-4 is a fluorescent compound with an excitation peak at 495 nm and an emission peak at 528 nm.

Mechanism

Calcium sensing

Fluo-4 has two important functional subunits, a BAPTA-based ionophore and a fluorescein-based fluorophore. While unbound to calcium, the BAPTA core quenches the fluorophore part of the probe through photoinduced electron transfer (PeT), thereby rendering Fluo-4 non-fluorescent. Upon binding Ca²⁺, PeT is reduced, enabling the fluorophore subunit to fluoresce, resulting in a more than 100-fold increase in fluorescence intensity.

Cell permeability

Cell permeability is enabled by attaching an acetoxymethyl (AM) ester group to Fluo-4. This increases its hydrophobicity and allows it to readily penetrate the intact membranes of live cells. Once inside, ubiquitous non-specifc intracellular estrases cleave the AM ester group. This is important, as the AM ester group quenches fluorescence. Therefore, only calcium in live cells, with active enzymes, will be detected.

Cellular retention

Organic-anion transporters located in cell membranes can cause Fluo-4 to exit the cell, leading to poor dye retention and increased background fluorescence. One solution is to utilize an inhibitor, such as probenecid. Alternatively, a probe with improved cellular retention, such as Cal-520 AM, can be used.

Applications

Fluo-4 AM can be used to study intracellular free calcium concentrations on a variety of instrumentation platforms, such as fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Given the prevalence of GPCRs as a target for therapeutics, Fluo-4 is also significant in high throughput screening and drug discovery applications.

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
mg	÷	1096.95 g/mol	=	mL	x	mM	=	mmol

Citations

View all 28 citations: Citation Explorer

Calcium Imaging in Brain Tissue Slices

Authors:

K{\'e}kesi, Orsolya and Keembiyage, Nisal and Buskila, Yossi

Suppressing ERp57 diminishes osteoclast activity and ameliorates ovariectomy-induced bone loss via the intervention in calcium oscillation and the calmodulin/calcineurin/Nfatc1 pathway.

Authors:

Yuan, Tao and Wang, Yi and Wang, Haojue and Lu, Qizhen and Zhang, Xin and Li, Ziqing and Sun, Shui

Journal:

Heliyon (2024)

Cytoplasmic calcium influx mediated by plant MLKLs confers TNL-triggered immunity

Authors:

Shen, Qiaochu and Hasegawa, Keiichi and Oelerich, Nicole and Prakken, Anna and Tersch, Lea Weiler and Wang, Junli and Reichhardt, Frowin and Tersch, Alexandra and Choo, Je Cuan and Timmers, Ton and others,

Journal:

Cell Host \& Microbe (2024)

Contrary effects of the gut metabolites deoxycholate and butyrate on the acetylcholine-evoked calcium response in an enteroendocrine cell model

Authors:

Pfanzagl, Beatrix and Jensen-Jarolim, Erika

Journal:

Endocrine and Metabolic Science (2024): 100167

Biological and mutational analyses of CXCR4--antagonist interactions and design of new antagonistic analogs

Authors:

Meng, Qian and Zhu, Ruohan and Mao, Yujia and Zhu, Siyu and Wu, Yi and Huang, Lina SM and Ciechanover, Aaron and An, Jing and Xu, Yan and Huang, Ziwei

Journal:

Bioscience Reports (2023)

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	0.16 ¹

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
CAS	273221-67-3

Instrument settings

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

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Page updated on September 27, 2025