Fluorochrome/protein

A similar method is used for beads with defined fluorochrome content (e g, Quantum beads, Flow Cytometry Standards Corporation), except that the beads are not subjected to the antibody-staining procedure The number of fluorochrome molecules per cell is deduced from the standard curve, and is converted to bound antibody molecules from knowledge of the fluorochrome protein ratio of the conjugate. 

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. 

A UV laser is needed for exciting the blue-fluorescing agents, 4, 6-diamidino-2-phenyhndole (DAPI) and Hoechst 33342, which are DNA-intercalating stains, and for indo-1, a fluorescent calcium chelator dye. Violet diode lasers that are offered in some newer instruments accommodate fluorochromes such as Cascade Blue, Pacific Blue, and cyan fluorescent protein, and are also capable of exciting DAPI (Shapiro and Perlmutter 2001 Telford et al., 2003). 

Calculation of Protein Concentration and Fluorochrome-to-Protein Ratio... 

The protein concentration P is determined by measuring the sample dissolved in PBS (reference PBS) at 280 nm with consideration to the fluorochrome-specific absorption at 495 nm ... 

The ratio F/P (moles fluorochrome per milligram of protein) is calculated by... 

The quantum efficiency of a given fluorochrome ultimately determines the sensitivity attainable. Thus protein fluorochromes derived from marine algae, such as phycoerythrin, have very high quantum efficiency in comparison to small chemical fluorochromes, such as fluorescein. For analysis of low antigen densities, phycoerythrin is to be preferred. 

Autofluorescence can be very useful for localizing proteins without staining or other perturbations of the product but it can be a major issue in the interpretation of stained dairy foods. It remains as background fluorescence in the presence of fluorochromes giving similar colors, particularly the protein dye, ANS and FTTC-conjugated molecules such as lectins. 

Although there are a number of important food components which are naturally fluorescent (e.g., cereal brans, lignified materials such as pea, soy and cotton fiber, and even proteins and pigments), detection of many food components requires application of specific fluorochromes or diachromes. Therefore, quantitative analysis using microscopic imaging also requires judicious use of sensitive dyes or stains suitable for visualization and rapid measurement. The dyes must be stable, non-toxic to liing cells, easily and inexpensively... 

The level of fluorescein modification in a macromolecule can be determined by measuring its absorbance at or near its characteristic excitation maximum (—498 nm). The number of fluorochrome molecules per molecule of protein is known as the F/P ratio. This value should be measured for all derivatives prepared with fluorescent tags. The ratio is important in predicting the behavior of antibodies labeled with FITC (Hebert et al., 1967 Beutner, 1971). Using the known extinction coefficient of FITC in solution at pH 13 (e498nm = 8.1-8.5 x 104 Jobbagy andjobbagy, 1972 McKinney et al., 1964), a determination of derivatization level can be made after excess FITC is removed. At pH 7.8, the absorbance of protein-coupled FITC decreases by 8%. 

Although many applications of flow cytometry involve the staining of cells for proteins expressed on the outer membrane, cells also have many proteins that are not displayed on their surface. With appropriate procedures, flow cytometry can provide a means to analyze these intracellular proteins. The outer cell membrane is impermeable to large molcules like antibodies however, if we intentionally fix cells to stabilize proteins and then disrupt the outer membrane, the cells can be stained with fluorochrome-conjugated monoclonal antibodies against intracellular proteins. After time to allow the antibodies to pass through the now-permeabilized membrane, the cells are washed to remove loosely bound antibodies and then are run through the flow cytometer to measure their fluorescence intensity. 

By way of a completely different type of functional analysis, we can look at the use of tracking dyes to label and follow cells. Cells can be stained with lipophilic fluorochromes or with fluorochromes that bind nonspecifically to all proteins. An example of the lipophilic fluorochromes are the so-called PKH dyes that insert themselves into the bilayer of cell membranes. The dye carboxyfluorescein succinimidyl ester (CFSE) binds to the free-amino groups on all cell proteins. These two types of dyes can be used to stain cells with considerable stability. The stained cells can then be injected into an animal and tracked to their homing location. 

Studies of stem cell progression towards the completely differentiated mature cells have already identified several intermediary precursors, organized in a cascade (Shizuru et al., 2005). The best known and studied cellular differentiation cascade is the hematopoietic system (Figure 20.3). Within hematopoiesis, it is possible to identify many intermediary precursors between the hematopoietic stem cell and mature blood cells. This identification is based mainly on the phenotypic profile of cellular surface proteins, using flow cytometry as the main tool. This is a relatively simple technique that involves coupling a monoclonal antibody (mAb) with a fluorescent marker (fluorochrome). In this way, diverse cellular markers can be combined and thus a cellular subpopulation can be defined, as shown in Figure 20.3. 

The ELISA method (in its various modifications), employing radionuclide markers (radioimmunoassay RIA), enzymatic markers (enzyme immunoassay EIA) (Dill et al., 2006), or fluorescent dyes (fluorophores or fluorochromes) (Hildebrand et al., 2005) (FEIA—fluoroenzyme immunoassay, Immuno-CAP-system) or nanoparticles (Dequaire et al., 2000) are commonly used as materials for protein labeling in an immunoassay. 

Conjugation of antisera with fluoroscein or rhodamine These are the two common fluorochromes and they are covalently linked to proteins by using activated forms (e.g. the isothiocyanates). 

---