Calcein, also known as fluorexon, fluorescein

Fluorescein

Fluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....

 complex, is a fluorescent dye

Dye

A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....

 with an excitation

Excited state

Excitation is an elevation in energy level above an arbitrary baseline energy state. In physics there is a specific technical definition for energy level which is often associated with an atom being excited to an excited state....

 and emission wavelengths of 495/515 nm, respectively. Calcein also self-quenches

Quenching (fluorescence)

Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on...

 even at concentrations below 100mM. It is used as a complexometric indicator

Complexometric indicator

A complexometric indicator is an ionochromic dye that undergoes a definite color change in presence of specific metal ions. It forms a weak complex with the ions present in the solution, which has a significantly different color from the form existing outside the complex.Complexometric indicators...

 for titration

Titration

Titration, also known as titrimetry, is a common laboratory method of quantitative chemical analysis that is used to determine the unknown concentration of an identified analyte. Because volume measurements play a key role in titration, it is also known as volumetric analysis. A reagent, called the...

 of calcium

Calcium

Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...

 ions with EDTA

EDTA

Ethylenediaminetetraacetic acid, widely abbreviated as EDTA , is a polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ligand...

, and for fluorometric determination of calcium. It has the appearance of orange crystals.

Applications

The acetomethoxy derivate of calcein (calcein AM) is used in biology as it can be transported through the cellular membrane into live cells, which makes it useful for testing of cell viability

Viability

Viable or viability is the ability of a thing to maintain itself or recover its potentialities.Viable or viability may also refer to:...

 and for short-term labeling of cells. An acetomethoxy group obscures the part of the molecule that chelates Ca²⁺, Mg²⁺, Zn²⁺ and other ions. After transport into the cells, intracellular esterases remove the acetomethoxy group, the molecule gets trapped inside and gives out strong green fluorescence. As dead cells lack active esterases, only live cells are labeled and counted by flow cytometry

Flow cytometry

Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...

.

Calcein is now rarely used as a Ca²⁺ or Mg²⁺ indicator because its fluorescence is directly sensitive to these ions only at strongly alkaline pH, and thus it is not particularly useful for measuring Ca²⁺ or Mg²⁺ in cells. Fluorescence of calcein is quenched strongly by Co²⁺, Ni²⁺ and Cu²⁺ and appreciably by Fe³⁺ and Mn²⁺ at physiological pH. This fluorescence quenching response can be exploited for detecting the opening of the mitochondrial permeability transition pore. Calcein is commonly used for cell tracing and in studies of endocytosis and gap junctions.

The acetoxymethyl ester of calcein is also used to detect drug interactions with multidrug resistance proteins (ABC transporters ATP-binding cassette transporter genes

ATP-binding cassette transporter genes

ATP-binding cassette transporters are members of a protein superfamily that is one of the largest and most ancient families with representatives in all extant phyla from prokaryotes to humans...

) in intact cells as it is an excellent substrate of the multidrug resistance transporter 1 (MDR1) P-glycoprotein

P-glycoprotein

P-glycoprotein 1 also known as multidrug resistance protein 1 or ATP-binding cassette sub-family B member 1 or cluster of differentiation 243 is a glycoprotein that in humans is encoded by the ABCB1 gene...

 and the :de:Multidrug Resistance-Related Protein (MRP1). The calcein AM assay can be used as a model for drug-drug interactions, for screening transporter substrates and/or inhibitors; and also to determine in vitro drug resistance of cells, including samples from patients.

Calcein is also used for marking freshly hatched fish

Fish

Fish are a paraphyletic group of organisms that consist of all gill-bearing aquatic vertebrate animals that lack limbs with digits. Included in this definition are the living hagfish, lampreys, and cartilaginous and bony fish, as well as various extinct related groups...

 and for labeling of bone

Bone

Bones are rigid organs that constitute part of the endoskeleton of vertebrates. They support, and protect the various organs of the body, produce red and white blood cells and store minerals. Bone tissue is a type of dense connective tissue...

s in live animals.