Calcein

Structural formula
General
Surname	Calcein
other names	Fluorexone Fluorescein complexon Fluorescein-2,7-bis (methyleniminodiacetic acid) 2,7-bis (( N , N -bis (carboxymethy) amino) methyl) fluorescein 4,5-bis (( N , N -bis (carboxymethy) amino) methyl) fluorescein Oftascein ( INN )
Molecular formula	C 30 H 26 N 2 O 13
Brief description	orange powder
External identifiers / databases
CAS number 1461-15-0 EC number 215-957-1 ECHA InfoCard 100.014.507 PubChem 65079 ChemSpider 58589 DrugBank DB11184 Wikidata Q413006
properties
Molar mass	622.51 g mol −1
Physical state	firmly
solubility	soluble in water and acids with yellow, in alkalis with orange to pink color sparingly soluble in water (4 g l −1 at 20 ° C) and ethanol (0.6 g l −1 ) Calcein-AM is soluble in dimethyl sulfoxide
safety instructions
GHS labeling of hazardous substances no GHS pictograms H and P phrases H: no H-phrases P: no P-phrases
As far as possible and customary, SI units are used. Unless otherwise noted, the data given apply to standard conditions .

Calcein , also known as Fluorexon , is a fluorescein - derivative and a fluorescent dye with an excitation maximum at 494  nm (excitation) and an emission maximum ( emission maximum ) of 517 nm (at a pH of 9.0). A concentration above 0.1 mol / l leads to fluorescence quenching .

structure

Calcein is a non-planar aromatic compound . As an anion, the various carboxy groups are deprotonated, so that calcein is hydrophilic under those conditions. Commercially it is available as the free acid and the 4 ', 5' isomer. However, various other isomers have been made commercially available over time.

Applications

Complexometry

Due to its numerous carboxylate groups, calcein is used in complexometry as an indicator for titrations of calcium ions with EDTA and for fluorometric determinations of calcium and magnesium. In addition, it also complexes other ions such as Cu ²⁺ , Fe ²⁺ , Fe ³⁺ and Ni ²⁺ .

Cell biology

After calcein-AM is taken up into the cell, it is converted to calcein by esterases (bottom). This can complex calcium ions, which leads to a green fluorescence. Since only living cells have sufficient esterase activity, only living cells fluoresce green after excitation.

As acetoxymethyl ester of calcein is calcein-AM (AM = A acetoxy m denotes ethyl ester). Four carboxy groups are esterified here, and it is also present as a lactone. The two remaining hydroxyl groups are esterified with acetic acid. Calcein-AM is used in cell biology . It is used to test cell viability ( cell adhesion , chemotaxis ) and for short-term marking of living cells. Alternatively, Fura-2 , Furaptra , Indo-1 and Aequorin can be used.

Calcein-AM can be transported across the cell membrane into living cells. The acetoxymethyl group masks the part of the molecule that is able to chelate calcium . After transport into the cell, the acetoxymethyl and acetate groups are enzymatically split off by esterases and converted into calcein. This is then able to bind (complex) calcium ions within the cell, which results in a strong, green fluorescence. Calcein is hardly cytotoxic as it does not affect vital cell processes such as cell division . The complex remains in the cell because it cannot pass through the cell membrane itself. However, many cells have an ABC transporter that actively removes calcein from the cell. These cells then also do not light up. The formation of these transporters is an important factor in the formation of resistance to drugs , for example cytostatics and antibiotics .

Since dead cells have no active esterases and therefore cannot produce calcium-binding calcein, only living cells are marked. These fluoresce green. Often a second fluorescent dye is also used, e.g. B. the ethidium homodimer-1 , which can penetrate through damaged cell membranes into the cell and there bind to nucleic acids . Dead cells are colored red and can be clearly differentiated from living cells. In another application it serves as a cell marker for apoptosis . Calcein can also be injected directly into the cells.

Outside the cell, calcein is used to mark freshly hatched fish or as a test substance in dermatology. The number of cells on microtiter plates can also be tested with calcein.

literature

• Horobin, RW. and Kiernan, YES. (2002): Conn's Biological Stains: A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine. BIOS Scientific Publ., 10th edition; ISBN 1-85996-099-5 ; P. 249ff.

Individual evidence

1. ↑ ^{a b} entry on calcein. In: Römpp Online . Georg Thieme Verlag, accessed on November 17, 2014.
2. ↑ Calcein data sheet at Panreac.com, accessed November 29, 2012.
3. ↑ interchim.fr: Calcein product information , accessed on November 17, 2014.
4. ↑ Calcein data sheet from Sigma-Aldrich , accessed on May 25, 2011 ( PDF ).
5. ↑ Horobin, RW. and Kiernan, YES. (2002): Conn's Biological Stains: A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine. BIOS Scientific Publ., 10th edition; ISBN 1-85996-099-5 ; P. 249.
6. ↑ Marking fry using calcein .