Flow cytometry labels

EIA was originally developed as a histological technique to localize specific ceUular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Eluorophore-labeled antibodies have also been used widely for flow cytometry appHcations using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

A variation on this method, called fluorescent in situ hybridization (FISH), uses fluorescent-labeled DNA and RNA probes for detection and visualization of single cells by microscopy or flow cytometry.7 80 The FISH technique is popular because of its sensitivity and speed of visualization fluorescent dyes can be used to produce probes with different colors for simultaneous detection of several organisms.76,81,82... 

Recently, Beatty and Tirrell [201] relied on the simultaneous or sequential addition of two reactive Met analogs, Aha and Hpg, to enable the fluorescent tagging of two protein populations within cells. The first demonstration of two-dye labeling of metabolically tagged cells was described in 2007 by Chang and co-workers [202], who used flow cytometry to show that cells treated with two reactive sugars could be labeled with distinct fluorophores. 

The synthesis and characterization of a somatostatin receptor-specific peptide H2N-(DPhe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, labeled with an indo-dicarbo- and an indotricarbocyanine dye at the V-terminal amino group were described in [34], The ability of these fluorescent contrast agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, wherein the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. The intracellular localization was visualized using NIR fluorescence microscopy. 

DCIA has been used to label numerous proteins and other biomolecules, including phospholipids (Silvius et al., 1987), to study the interaction of mRNA with the 30S ribosomal subunit (Czworkowski et al., 1991), in the investigation of cellular thiol components by flow cytometry (Durand and Olive, 1983), in the detection of carboxylate compounds using peroxyoxalate chemiluminescence (Grayeski and DeVasto, 1987), and for general sulfhydryl labeling (Sippel, 1981). 

J. A. Steinkamp and H. A. Crissman, Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry, Cytometry 14, 210-216 (1993). 

A fluorescence-activated cell sorter (FACS) is a flow cytometry instrument used to separate and identify cells in a heterogeneous population. Cell mixtures to be sorted are first bound to fluorescent dyes such as fluorescein or phycoerythrin. The labeled cells are then pumped through the instrument and are excited by a laser beam. Cells that fluoresce are detected, and an electrostatic charge is applied. The charged cells are separated using voltage deflection. 

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so on) can be used to detect or measure any cell constituent, provided that the tag reacts specifically and stoichiometrically with the cellular constituent in question (2). Today, the repertoire of fluorescent probes is expanding almost daily see Chapter 14). One area that has benefited from the ever-increasing number of fluorescent probes is flow cytometry. 

Tim, E. A. and Stewart, C. C. (1992) Eluorescence in situ hybridization in suspension (FISHES), using digoxigenin-labeled probes and flow cytometry. Biotechniques 12, 362-367. 

A limitation of the flow cytometric binding assay has been the precise determination of the receptor affinity and calculation of the receptors per cell. This limitation appears to have been overcome by the development of fluorescein and phycoerythrin compensation-calibration standards (Flow Cytometry Standards Corp., Research Triangle Park, NC). These standards have made it possible to quantify the fluorescence intensity of samples labeled with fluorescein or phycoerythrin, and relate the intensity to molecules of equivalent soluble fluorochrome. These standards have been utilized in quantitative studies of neutrophil chemoattractant-ligand interaction (4). 

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