Fluo-4 AM

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.