Analytical techniques flow cytometry

The 30-year history of MAbs has been a rollercoaster ride to success. From hype to depression, and back to hype, is probably the shortest summary of what has happened. MAbs have been rapidly introduced into a number of applications within and outside the medical field (Table 1.2) [52-54]. Analytical in vitro methods such as enzyme-hnked immunosorbent assay (ELISA), radioimmunoassay (RIA), various blotting techniques, flow-cytometry, immunofluorescence, confocal imaging, and im-munohistochemistry are each dependent upon the use of polyclonal or monoclonal antibodies. 

Special emphasis is placed on the carbohydrate-mediated cell - target system interaction by describing hints and pitfalls of assays for cytoadhesion, specificity, cytoinvasion, and cytoevasion. In addition, basic considerations are presented to discriminate between active and passive uptake as well as to detect lysosomal accumulation. Finally, the pros and cons of two useful analytical techniques, namely, flow cytometry and confocal laser scanning microscopy, are described in detail. 

Abstract Flow cytometry is a technique for rapidly examining multiple characteristics of individual cells, by recording fluorescence signals emitted from cell-associated reporter molecules, and measuring cellular light scattering properties. This chapter introduces the principles and practice of flow cytometry, and reviews examples from the literature that highlight applications of this experimental tool in the neurosciences. The chapter concludes with protocols for three basic procedures that illustrate some practical aspects of analytical flow cytometry. 

Analytical flow cytometry offers a rapid and facile means of monitoring cellular receptor content. For example, multiparameter flow cytometry techniques were used to monitor expression of GABAa receptor subunits during neurogenesis in embryonic rat brain (Marie et al., 2001). The content of the cell surface p75 neurotrophin receptor was measured in a heterogeneous population of mouse dorsal root sensory neurons, from which high and low p75 subsets were subsequently isolated by cell sorting (Barrett et al., 1998). 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Chemical cytometry is a highly specialized class of analytical techniques in which the contents of individual biological cells are evaluated by chemical separations (e.g., capillary electrophoresis). In contrast to the more conventional technique of flow cytometry, in which intact cells are evaluated for the presence of one or a few markers, in chemical c)flometry, each ceU to be analyzed is lysed, such that (theoretically) all of the chemical constituents can be detected, identified, and quantified. 

The techniques that are currently used by most laboratories to measure cyt c release include ELISA, Western blot, and flow cytometry (Kim et al., 2007 Ott et al., 2002 Christensen et ah, 2013 Adachi et al., 2004). Despite providing high sensitivity and selectivity, these traditional analytical methods stiU have some drawbacks, such as time-consuming, sophisticated, expensive equipment, limitations in colored sample analysis, and the demand for skilled professionals. To minimize limitations imposed by traditional methods, electrochemical biosensors/immunosensors combined with the high specificity of conventional methods also present several advantages including the possibility of point-of-care testing development. 

Like antibodies, aptamers are characterized by very impressive, unsurpassed affinity, and selectivity properties. Such remarkable feature has determined immense potentialities in the diagnostic field and various analytical systems have been developed, notably in the field of biosensors, ELISA-type assays, flow cytometry, or separation techniques [6, 7]. Specifically, aptamers constitute, with antibodies, the most popular affinity reagent employed for the development of bioaffinity-based LC and CE methodologies. However, the aptamers present many advantages over antibodies. Aptamers can be regenerated within minutes via a denaturation-renaturation... 

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