Proliferation Assay techniques

Characteristic of immune function evaluations, there is considerable diversity in the approaches to proliferation assays, from the experimental design to the analytical method. Experimental procedures necessitate sterile technique and cell culture expertise to ensure accurate assessment of the cellular response to a particular stimulant. Culture conditions vary depending on the cell source and type of stimulant, but generally are conducted at 5% CO2,37 °C for 48 to 96 hours. The source of lymphocytes may be from peripheral blood, spleen. 

A variety of techniques have been employed to assess the immune response to beryllium. Ho vever, the ease, sensitivity, and specificity have made the lymphocyte proliferation assay using H-thymidine the standard technique (Rossman et al. 1988, Mroz et al. 1991). In the past, this technique has been referred to as the lymphocyte transformation test (LTT), but transformation is an older term that is no longer applicable. The cells respond by proliferating (a normal process) and are not transformed, which today implies a malignant process. Hence, the test is currently known as the lymphocyte proliferation test (LPT). 

Fig. 7 a The cumulative release of VEGF from PLGA scaffolds fabricated by a gas foaming particulate leaching technique, b The bioactivity of VEGF released from these matrices at days 2, 7, and 14 was comparable to the effect obtained with known doses (5 and 20 ng/mL) of VEGF not incorporated into matrices (determined by an endothelial cell proliferation assay) (J. Control. Release [78], with permission of Elsevier)... 

Incubation periods in excess of 2 h were required before this activity was detected in cell-free supernatants. More recently, the use of cDNA probing of Northern transfers (to detect specific mRNA levels), the use of ELISA techniques (to detect protein levels immunologically) and the development of more specific bioassays (culture techniques in which a biomolecule stimulates proliferation in a particular cell line) have resulted in a more thorough analysis of IL-1 production by neutrophils. IL-1 is only poorly expressed in blood neutrophils because mRNA for this cytokine is detectable only at very low levels (if at all), and protein production is usually below the level of detection of most assays. However, exposure of neutrophils to lipopolysaccharide (LPS), or to cytokines such as GM-CSF, TNF or IL-1 itself, results in a rapid but transient increase in IL-1 expression. 

Cell proliferation can be measured by different techniques in various in vitro models and is used as one of the most specific endpoints for DNT evaluation. A commonly used assay is the bromo-deoxyuridine (BrdU) incorporation where analogues nucleotides are integrated into newly synthesized DNA permitting indirect detection of rapidly proliferating cells by fluorescent labelled anti-BrdU antibodies [49], Cytotoxicity assays, such as MTT and ala-marBlue, are also sometimes used to measure proliferation [49]. However, these assays do not distinguish between cell death and inhibited cell proliferation which is of particular importance in DNT studies. 

Cell-Based Bioassay For a cell-based Nab bioassay, treated cells respond directly or indirectly to the drug in a concentration-dependent manner. Possible biological responses of the cells to chug treatment include cell proliferation, apoptosis, phosphorylation, chemokine release, and expression of proteins or genes [15,22,28 30]. These responses may by quantitated by techniques such as immunoassay, multiplex assays [31], flow cytometry [23], and gene expression profiling [32]. 

Messele T, Roos MT, Hamann D, Koot M, Fontanet AL, Miedema F, ScheUekens PT, Rinke de Wit TF. Nonradioactive techniques for measurement of in vitro T-ceU proliferation alternatives to the [(3)H]thymidine incorporation assay. Chn Diagn Lab Immunol 2000 7 687-692. 

Bioassays are frequently used as an alternative or in addition to immunoassay techniques. Bioassays, in contrast to immunoassays, quantify not the pharmacologically active substance, but its biological activity, for example, in cell culture models based on cell differentiation, cell proliferation, or cytotoxicity as well as gene expression assays or whole animal models. Frequent major problems with bioassays comprise a high variability in the measured parameters, lack of precision, and their time- and labor-intensive performance. Furthermore, bioassays oftentimes also lack specificity for the measured compound, as they may also detect the response to bioactive metabolites [16,17]. 

Besides ligand-induced signaling, other apphcations of this assay are the measurement of cell proliferation or cell viability (Kho et al., 2015). Chemotaxis can be measured in real time by combining this technique with the chemotaxis assay described in Section 3.2.6. Cells in the upper chamber of a CIM-plate migrate through the porous membrane into the chemoattractant-containing lower chamber and adhere to the microelectrode embedded in the bottom of the plate, leading to an increase in impedance (Iqbal et al., 2013). 

The yeast two-hybrid system is a powerful genetic technique that has been used successfully to study protein-protein interactions in vivo from a wide variety of biological sources, including yeast, plant, and mammalian (Fields and Song, 1989). The two-hybrid system provides a sensitive method to detect relatively weak and transient protein-protein interactions that may not be biochemically detectable. The principle of this assay is based on the reconstitution of transcriptional factors that promotes the expression reporter genes, which then allow the proliferation of yeast under restrictive conditions. Using the two-hybrid system, we successfully isolated IPCEFl and a novel kinesin motor proteins from a rat brain cDNA library as interactors of cytohesin 2 ARF GEF and centaurin-al ARF GAP, respectively (Venkateswarlu, 2003). The procedure used to map the binding domains of cytohesin 2 and IPCEFl, and to characterize the cytohesin 2 and IPCEFl selectivity is outlined here. 

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