Flow cytometry using fluorescently-labeled

Intracellular cytokine levels (Flow cytometry using fluorescent labels)... 

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... 

These methods use fluorescent labels, such as propidium iodide, ethidium bromide, or DAPI (4, 6 -diamidino-2-phenylindole), which are incorporated into the DNA, allowing chromatin condensation and nuclear fragmentation to be visualized under a microscope with the appropriate fluorescence filters. To allow fluorochromes to enter the cells and reach the nucleus, the cells need to be prepermeabilized, for example, with 70% ethanol at -20°C. LMW-DNA fragments may be lost by the permeabilization, decreasing the amount of DNA inside the cells. The lower nucleic acid concentration results in a lower fluorescence intensity in apoptotic cells, which can be detected by fluorescence microscopy or flow cytometry (Calle et al., 2001). 

Flow cytometry is a method in which cells flow one-fay-one through an area illuminated fay a laser beam. Information about the cells is obtained using fluorescent labels. Die development of hi i-affinity DNA stains allows DNA fragment siang using flow technology. Die basic idea is that the amount of (fye bound 1 the DNA fragments is... 

Fig. 4 Metabolic DNA labeling using alkyne- or alkene-modified nucleosides. A 5-Ethynyl-2 -deoxyuridine (EdU) [153], (2 S)-2 -deoxy-2 -fluoro-5-ethynyluridine (F-ara-EdU) [160] and 5-vinyl-2 -deoxyuridine (VdU) [167], B Labeling strategy cells are incubated with the alkyne-modified nucleoside EdU, which is incorporated into the cellular genome in proliferating cells. Subsequently, the cells are fixed, permeabilized and components for the Cu -mediated azide alkyne cUck reaction with flumescent azides are added. Detection can be carried out by flow cytometry or fluorescence microscopy...

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). 

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. 

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. 

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