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Fluo-4 AM

Ultrapure Grade; CAS 273221-67-3
Fluo-4 AM is a cell permeable, green fluorescent calcium probe used in fluorescence microscopy and flow cytometry.

Background

Fluo-4 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 (Kd 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.

Calcium imaging protocol

Quick summary

  1. Prepare stock solution (2 to 5 mM)
  2. Prepare working solution (2 to 20 µM)
  3. Add working solution to cell plate
  4. Incubate at 37 °C (30 to 60 minutes)
  5. Replace working solution to remove excess probes
  6. Measure fluorescence (Ex/Em = 490/525 nm)

Full protocol

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

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Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
Fluo-3, AM *CAS 121714-22-5*50651586,00010.151
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*50651586,00010.151
Fluo-3, AM *Bulk package* *CAS 121714-22-5*50651586,00010.151
Fluo-3FF, AM *UltraPure grade* *Cell permeant*50651586,00010.151
Fluo-8H™, AM495516234300.161
Fluo-8L™, AM495516234300.161
Fluo-8FF™, AM495516234300.161
Fluo-5F, AM *Cell permeant*494516--
Fluo-5N, AM *Cell permeant*494516--
Rhod-4™, AM523551-0.11
Show More (1)

Citations

View all 27 citations: Citation Explorer
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)
P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2
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
Page updated on September 5, 2024

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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-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

CAS

273221-67-3

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