Detectability fluorochrome labels

Figure 9.8 Evaluation of binding of biopharmaceutical X to cynomolgus monkey peripheral blood cells. Cynomolgus monkey cells were stained with fluorochrome-labeled biopharmaceutical X or the natural ligand for the human receptor. Expression of the target receptor on the cynomolgus cells was detected using antibodies specific for the target receptor.

Fluorochrome labeling of streptavidin or antibody Conjugation procedures should yield optimal fluorochrome/protein (F/P) ratios. Most economically, the desired F/P ratio is regulated by the initial weight of dye in the reaction mixture and the reaction is allowed to go to completion. Alternatively, with relatively more dye, the reaction is interrupted after a specific incubation period. The efficiency of labeling depends on the protein, the protein concentration, the specific fluorochrome and the purity of the fluorochrome (some preparations only 30%). Over- or undercoupling leads to nonspecificity or low detectability, respectively. 

Fig. 6. Activation of caspases detected by the fluorochrome-labeled caspase (FLICA) inhibitors assay. FiL-60 cells were untreated (A), treated in culture with camptothecin to induce apoptosis (B) (ref. 26). The cells were then electrostatically attached to microscope slides, incubated with staining solution of FAM-VAD-FMK as described in the protocol, and their green fluorescence (integrated value and pixel of maximal intensity) measured by LSC. Note the appearance of apoptotic cell subpopulation characterized by the increased green fluorescence (above the marked threshold level of the maximal pixel) reflecting activation of caspases that bind FAM-VAD-FMK.

This chapter deals with the detection of antigens that are accessible on the surface of isolated living cells using fluorochrome labels. By incubating live cells at 4°C, to prevent endocytosis of bound molecules, the attached antibody can remain on the cell surface, and either be observed in the live state or subsequently fixed. This method yields the greatest sensitivity and best morphologic preservation for detection of surface molecules using antibodies for microscopy. 

Fig. 1. Double immunofluorescence detection of cytoplasmic cytokeratin 19 intermediate filaments (CK19) (FITC, green/ filamentous) and the cell-membrane associated connexin 43 (Cx43) gap junction protein (Cy3, red/dot-like) in the stratified squamous epithelium of human cervix. Combination of mouse monoclonal (CK19) and rabbit polyclonal (Cx43) antibodies and distinct fluorochrome-labeled secondary antibodis. Confocal laser scanning microscopy, a projection of five optical layers.

Chromosomal in situ hybridization enables determination of the presence and location of DNA sequences complementary to a labeled probe along chromosomes and within interphase nuclei. The use of directly fluorochrome-labeled probes means that sites of probe hybridization can be directly visualized. Lengthy detection procedures, as in the indirect immunochemistry methods to detect biotin or digoxigenin, are not needed. However, because there is no signal amplification, sensitivity is limited and the system is used mostly to detect middle- to high-copy number (Fig. 1) or pooled probes, although antibody amplification of some fluorochrome labels is possible (1), and then the method must be evaluated in comparison with digoxigenin (Chapter 27) or biotin (Chapter 25). 

Fluorochromes are visualized by excitation with light of one (excitation) wavelength and imaging emitted fluorescence at another (emission) wavelength using appropriate filters. The properties and methods of visualization of the fluorochromes fluorescein and rho-damine, which are used to detect digoxigenin, are the same as for direct fluorochrome-labeled probes and are outlined in Chapter 26. 

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.

TUNEL stands for terminal deoxynucleotidyl transferase x-dUTP nick end labeling. This assay is based on the detection of DNA fragments marked by an enzyme that incorporates modified nucleotides to the 3 -OH ends of the fragments, which can be then specifically detected. The enzyme is a deoxynucleotidyl transferase, which can act in absence of a complementary strand. Among the nucleotides, there is one specifically marked with a fluorochrome, an enzyme, or an antigen. This allows different methods of detection. 

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). 

As shown in Figure 12.6, two types of assays are often used in analyzing possible protein interactions and evaluating protein expression levels direa and indirect (or sometimes called sandwich) assays. Direct assays typically involve a target protein that are prelinked onto an assay platform such as a well-plate array or a cover slip. Subsequently, detection of the target protein is carried out with a primary antibody that is labeled with a fluorochrome. On the other hand, more target-specific sandwich assays typically start with prelinking of an unlabeled primary antibody to... 

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