Fluorescence activated cell analysis

Nolan, G. P., Fiering, S, Nicolas, J. F., and Herzenberg, L A. (1988) Fluorescence-activated cell analysis and sorting of viable mammalian cells based on p-D-galactosidase activity after transduction of Escherichia coli lacZ Proc. Natl Acad. Sci. USA 85, 2603-2607. 

The chemical composition of particles can be just as varied as their shape. Commercial particles can consist of polymers or copolymers, inorganic constructs, metals and semiconductors, superparamagnetic composites, biodegradable constructs, and synthetic dendrimers and dendrons. Often, both the composition of a particle and its shape govern its suitability for a particular purpose. For instance, composite particles containing superparamagnetic iron oxide typically are used for small-scale affinity separations, especially for cell separations followed by flow cytometry analysis or fluorescence-activated cell sorting (FACS). Core-shell semiconductor particles, by... 

Fluorescence-activated cell sorters (FACS) have been used to separate subpopulations of cells for subsequent treatment or analysis (see Chapter 30). However, this approach requires that the cells be in suspension. In the case of adherent cells, some cannot be easily suspended, or the treatments used to suspend them may interfere with subsequent analysis. In these situations, a laser microheam system capable of fluorescence imaging can serve two purposes. At low power, the laser can excite fluorescence to produce an image, and at a higher power, the laser can be used to kill the undesired cells. 

Sklar, L. A. and Finney, D. A. (1982) Analysis of ligand-receptor interactions with the fluorescence activated cell sorter. Cytometry 3,161-165. 

Newer strategies for stem cell identification have been developed based on the knowledge of cell functions. A primitive and multipotential subpopulation of bone marrow mononuclear cells has been identified on the basis of the intracellular presence of aldehyde dehydrogenase (ALDH). Those cells can be marked on the basis of the presence of ALDH and are called aldehyde dehydrogenase-bright cells (ALDH cells), allowing for their separation from a bone marrow aspiration mononuclear subpopulation under fluorescence-activated cell sorter (FACS) analysis. 

The various organs of the immune system such as spleen, lymph nodes, thymus and bone marrow containing the cells involved in the various immune responses offer the possibility to harvest these cells and perform in vitro assays for evaluation of effects on the immune system. When part of an in vivo animal study this may indicate a direct toxic effect of pharmaceuticals, that is, immunosuppression (Table 18.2). So, it is feasible to obtain cell suspensions for further evaluation such as determination of cellular subsets of T and B leukocytes by fluorescent activated cell sorter analysis (FACS analysis), and determination of natural killer (NK) cell activity of the spleen cell population. An advantage of this approach is that it may lead to identification of a biomarker to be used in clinical studies. In addition, in vitro stimulation of spleen cells with mitogens activating specific subsets may indicate potential effects on the functionality of splenic cell populations. Concanavalin A (Con A) and phytohemagglutinin (PHA) activate Tcells, while lipopolysaccharide (LPS) activates primarily B cell populations. Blood is collected for total white blood cell (WBC) determination and blood cell differential count. In addition, serum can be obtained for determination of serum immunoglobulins. 

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

Pel, R., Ploris, V., and Hoogveld, H. (2004a). Analysis of planktonic community structure and trophic interactions using refined isotopic signatures determined by combining fluorescence-activated cell sorting and isotope-ratio mass spectrometry. Freshw. Biol. 49, 546—562. 

---