Fluorescence flow cytometry

Viability-based technologies Direct epifluorescent filter microscopy Membrane laser scanning Fluorescence cytometry Fluorescence flow cytometry... 

Talyzina N. M., Moldowan J. M., Johannisson A., and Fago F. J. (2000) Affinities of early Cambrian acritarchs studied by using microscopy, fluorescence flow cytometry and biomarkers. Rev. Palaeobot. Palynol. 108(1-2), 37-53. 

Fay,S P., Posner, R G,Swann, W. N., and Sklar, L. A. (1991) Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry Biochemistry 30,5066-5075. 

Murphy, R. F. Powers, S. Cantor, C. R. Endosome pH measured in single ceUs by dual fluorescence flow cytometry rapid acidification of insulin to pH 6. J. CeU Biol. 1984, 98, 1757-1762. 

Zola H, Flego L, Sheldon A (1992) Detection of cytokine receptors by high-sensitivityimmuno-fluorescence/flow cytometry. Immunobiology 185 350-365 Review Article 58 Refe... 

Vergne, I. Constant, P Laneelle, G. Phagosomal pH determination by dual fluorescence flow cytometry. Anal. Biochem. 1998, 255, 127-132. 

Quantification of cell-to-cell communication. Flow cytometry meikes it easy to quantify cell-to-cell communication. As an example work of Kavanagh may be cited. Cells are scrape-loaded with Lucifer yellow with or without rhodamine labelled dextran. The transfer of the two dyes between donor and recipient cells can be studied easily using two colour fluorescence flow cytometry. 

The similar results have been obtained in [12] for 1 1 cage complex of 540 with encapsulated pyrene-R molecule. In this case, fluorescent flow cytometry suggested more intensive fluorescence... 

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). 

We will first summarize the fluorescence and spectroscopic assays that have been developed for the fluorometer and then describe their applications using flow cytometry. We will summarize research which exemplifies the utility of simultaneous measurement of responses and shows how these methods have provided Information about the signal transduction pathways and activation in neutrophils. 

Fig. 3. Flow cytometry analysis. CellQuest software. The fluorescence of 50,000 cells is measured.

In addition to these time-honoured methods, newer techniques involving bio-luminescense, fluorescent dyes (epifluorescence) and physical methods such as impedance, calorimetry and flow cytometry have been developed. A feature being sought in these methods is rapidity see section 5.6. 

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... 

Gunasekera,T. S. Veal, D. A. Attfleld,P. V. Potential for broad applications of flow cytometry and fluorescence techniques in microbiological and somatic cell analyses of milk. Int. J. Food Microbiol. 2003, 85, 269-279. 

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. 

Fig. 11.7 Effect of HU on ml-CAM-1 expression in the TrHBMEC (a) and EA-hy 926 (b). These cells were incubated with HU 250 pM for 48 h with or without 100 U mb of TNFaand IFNy. mlCAM-1 cellular expression was analyzed by flow cytometry. Results are the Mean Fluorescent Index (MFI) of one representative experiment, with overall trend in three other independent experiments being comparable. Parallel estimation of slCAM-1 release in the culture supernatant of TrHBMEC cells (6 independent experiments) revealed that without cytokines sICAM-1 was not detectable in the supernatant for the basal conditions. The results of HU-treated cells (c) in the presence of cytokines showed a significant increase in release of slCAM-1 (p <0.05).

Cellular uptake of MTX in an MTX-LDH hybrid was quantified by flow cytometry with FITC-conjugated MTX (MTX-FTIC). Human osteosarcoma (HOS) cells (5 x 105/1 ml) were treated with 50 pg/mL of MTX-FITC and 112 pg/mL of MTX-FITC-LDH, where the concentration of MTX-FITC in MTX-FITC-LDH was adjusted to maintain an equivalent amount of free MTX-FITC treated. After 1, 2, and 5h, cellular uptake was about 55-fold higher for MTX-FITC-LDH than for free MTX-FITC, as determined by the remarkably high fluorescence intensity of MTX-FITC-LDH compared to free MTX-FITC (Figure 13.6). This is evidence that MTX delivery in cells is greatly enhanced by using the MTX-LDH hybrid. 

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