Calcein, Fluorescein

Discussion. This method is based upon the formation of a fluorescent chelate between calcium ions and calcein [fluorescein bis(methyliminodiacetic acid)] in alkaline solution.29 The procedure described below30 has been employed for the determination of calcium in biological materials.31 ... 

Cadmium liquid amalgam 412 Cadmium sulphate, thermal analysis 498 Calcein (fluorescein iminodiacetic acid) ... 

Cadion, [l-(4-nitrophenyl)-3-(4-phenylazophenyl)-triazene] Calcein, Fluorescein Complexon Calcium hydride, in purification Carbon Black... 

Other Names Fluorescein, 2, 7 - w[[ (carboxymethyl)amino]melliyl]- Spiro[isobenzofuran-l (3H),9 -[9Fl]xantliene], glycine derivative 2,7-fi [iV,lV-to(carboxymethyl)aminomethylene]-fluorescein Acetic acid, [(3, 6 -dihydroxy-2, 7 -fluorandiyl)fci>(methylenenitrilo)]telra- Calcein Fluorescein complexon Fluorexon NSC 298193 Oftasceine CA Index Name Glycine, A7V -[(3, 6 -dihydroxy-3-oxospiro[isobenzofiiran-l(3II),9 -[9ir xanthene]-2, 7 -diyl) M(methylene)] is[N-(carboxymethyl)-CAS Registry Number 1461-15-0 Merck Index Number Not listed Chemical Structure... 

The quantification of fluorescent particles in cellular systems is difficult because several aspects such as autofluorescence, bleaching (see below), and quenching hamper analysis. Keep in mind that many fluorophores show a pH-dependent change in emission spectrum and intensity fluorescein-labeled dextrans (FITC-dextran) and calcein are strongly quenched upon acidification. If available, one should read the fluorescence intensity at its isosbestic point, where the intensity is not pH dependent. 

Calcein [2, 7 -bis- A, A -di(carboxymethyl)aminomethyl fluorescein] sodium salt [1461-15-0] M 666.5. Dissolve in distilled H2O and acidify with dilute HCl to pH 3.5. Filter off the solid acid and wash well with H2O. Redissolve ca lOg in 300ml H2O containing 12g of NaOAc. Ppte again by adding HCl, filter and wash with H2O. Add the solid to 200ml of EtOH stir for Ih and filter. Repeat the EtOH wash and dry the bright yellow solid in a vacuum. This acid decomposes on heating at ca 180°. See below for the prepn of the Na salt. [AC 28 882 1956]. 

Some analytes, such as riboflavin (vitamin B2)16 and polycyclic aromatic compounds (an important class of carcinogens), are naturally fluorescent and can be analyzed directly. Most compounds are not luminescent. However, coupling to a fluorescent moiety provides a route to sensitive analyses. Fluorescein is a strongly fluorescent compound that can be coupled to many molecules for analytical purposes. Fluorescent labeling of fingerprints is a powerful tool in forensic analysis.17 Sensor molecules whose luminescence responds selectively to a variety of simple cations and anions are available.18 Ca2+ can be measured from the fluorescence of a complex it forms with a derivative of fluorescein called calcein. 

By introducing the complexing moiety —CH2N(CH2C02H)2 into fluorescein or umbelliferone metallochromic indicators termed calcein or fluorexone, and calcein blue or umbellicomplexone are formed. These indicators exhibit green (and blue) fluorescence in dilute alkali which disappears at pH 12 but reappears on the addition of calcium, strontium, or barium. Problems arise from the difficulty of freeing the indicators from fluorescent impurities, from self-fluorescence, and from the quenching effect of copper ions. 

FIGURE 4.25 Sequences of nine consecutive pressure pulse injections with 4-s delay using a mixture of 38 iM calcein and 19 iM fluorescein (a). Species B, C, and D are derived from calcein. The durations of pressure pulse are (a) 0.3, (b) 0.35, and (c) 0.4 s [314]. Reprinted with permission from the American Chemical Society. 

Successful liquid-phase separation on-chip was first carried out in CE, because EOF pumping can be easily achieved in the microscale. For instance, six fluorescein-labeled amino acids are separated by CE on a Pyrex glass chip (10-pm-deep and 30-pm-wide channel) (see Figure 6.5). Separation was achieved in a very short time of about 15 s [324]. Similar CE separation of calcein and fluorescein were also reported [582]. Separation of a binary mixture of rhodamine B and dichlorofluorescein was even achieved in only 0.8 ms using a short separation length of 200 pm [604]. 

More recent staining procedures largely use fluorescent dyes to characterize the physiological and biochemical states of cells. Fluorescein Diacetate (FDA), a non-polar substance which crosses the membrane and is hydrolyzed by intracellular esterases in viable cells to produce fluorescein, exhibits yellow-green fluorescence when excited at 490 nm. Damaged or non-viable cells in general are unable to hydrolyze FDA or to retain fluorescein within the cell [172,173]. In combination with Ethidium Homodimer or Propidium Iodide, a similar esterase substrate, calcein acetoxy methyl ester (CAM) has been found to be reliable for viability assessment of protozoans, but not on Candida yeast, neither on bacteria such as Bacillus cereus and Escherichia coli [174]. 

Calcein solution Calcein (2, 7 -[(bis[carboxymethyl]-amino) methyl]-fluorescein)(Sigma- Sigma Chemical Co. (St. Louis, MO, USA) ( rccNote 7). Stock solutions of calcein (80 mM) are made by quickly dissolving solid calcein at pH 9.0 and then adjusting the pH to 7.5. 

Calcein (Fluorexone, Fluorescein Complexone) (formula 4.22), used as reagent for the determination of calcium, contains iminodiacetic acid groups in positions 2 and 7, and only one hydroxyl group in position 3. In the case of Calcein (like Xylenol Orange), the nitrogen atom of the iminodiacetic acid group participates in bonding the metal ion. 

Calcein sodium salt [2, 7 -bis- V,V-di(carhoxymethyl)aminomethyl fluorescein Na salt, Fluorexon, Fluorescein Complexon] [108750-13-6 diNa salt, 1461-15-0 free acid] M 666.5, pKEst(i) 1-9, pKesi<2) 2.5, pKEst(3) 8,0, pKesK4) 10.5 (all for N-CH2COOH), and pKes s) ... 

FIGURE 45.1 First published microchip electropherogram using LIF. A mixture of fluorescein and calcein (concentrations shown) were injected for 30 s across a separation channel and subsequently electrophoresed past the detector. (Reprinted from Harrison, D.J., et al., Analytical Chemistry, 64,1926,1992. Copyright 1992. With permission from American Chemical Society.)... 

Figure 15.11. Calcein AM and fluorescein dextran staining, used to depict cell viability and membrane permeability, respectively. Cells receiving only irradiation maintained cell viability and membrane integrity (a and c), whereas cells irradiated with nanoshells possessed circular zones of cell death and increased membrane permeability (b and d). Republished with permission from Ref. 43. Copyright 2003 National Academy of Sciences, USA.

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