Labeling fluorochromes

Most anionic FITC-labeled fluorochromes microinjected into the cytoplasm are compartmented by the plant cell vacuoles at rates that depend on their molecular size (14,15). 

Immunophenotype The process of identification and quantitation of cellular antigens through fluorochrome-labeled monoclonal antibodies. 

The nonfluorescent monofunctional azulenyl squaraine labels NIRQ700 an<3 NIRQ7so with absorption maxima between 600 and 750 nm are capable of quenching a number of NIR fluorochromes. They were specifically developed to extend the spectrum for FRET-based assays [100, 101]. 

Duijndam, W.A.L., Wiegant, J., Van Duijn, P., and Haaijman, J.J. (1988) A simple method for labeling the carbohydrate moieties of antibodies with fluorochromes. J. Immunol. Meth. 109, 289-290. 

Hopman AHN, Ramaekers FCS, Speel EJM (1998) Rapid synthesis of biotin, digoxigenin, trinitrophenyl, and fluorochrome labeled tyramides and their application for in situ hybridiza tion using CARD amplification. J Histochem Cytochem 46 771 777 Ino H (2003) Antigen retrieval by heating en bloc for pre fixed frozen material. J Histochem Cytochem 51 995 1003... 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

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). 

If antibodies conjugated to fluorochromes are not desirable, enzyme-labeled antibodies can also be used. In this case, in the presence of a substrate and a chromagen, an enzyme provides the indicator system necessary to visuahze the location of the antibody (Carson, 1997). Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the enzymes most commonly conjugated to antibodies not labeled by... 

Prior to coupling, prepare a gel-filtration column to separate the labeled antibody from the free fluorochrome after the completion of the reaction. The size of the column should be 10 bed volumes/sample volume see Note 1). 

The extent to which multiple fluorescence signals can be distinguished is demonstrated by resolution of five separate fluorochromes within individual, living 3T3 cells (24). None of the fluorochromes employed in this work was conjugated to an antibody, but the potential for extensive multiple labeling with a suitable set of fluorochromes and an appropriately equipped microscope system is clear. 

In this method, the primary antibodies are combined into a single cocktail and applied simultaneously in a single-reaction step. The same is done for the labeled secondary antibodies. Antibodies of different animal species or immunoglobulin isotypes (noncrossreactive) or autologous antibodies that are directly coupled to different enzymes/fluorochromes are required. Immuno-fluorescent methods are particularly suited for this method. 

If iV-formyl peptide chemoattractants conjugated with fluorochrome are purchased from a commercial source, the reagents listed in Subheading 2. referencing this note are not needed. Although this chapter has focused on iV-formyl peptides, other chemoattractants, such as complement-derived C5a, have also been fluorescently labeled and evaluated in flow cytometric assays (9-11). 

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). 

Fig. 3. Fluorescence profiles of 2, 7 -dichlorofluorescin-loaded cells assayed in whole blood. (A) Compares the fluorescence histograms of unstimulated, control cells (shaded curve) with granulocytes exposed to opsonized S. aureus (open curve). (B) illustrates the two-color analysis profde of the granulocytes that were exposed to Texas Red-labeled S. aureus. Red fluorescence is the result of particle association with each granulocyte, whereas green fluorescence is the result of the oxidation of 2, 7 -dichlorofluorescin to 2, 7 -dichlorofluorescein (DCF). The red and green fluorescence analyses were performed with log-scale detection amplification for each fluorochrome.

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