Cell-based assay protocol

Here are a few textbooks that cover a number of the specific hormone signaling systems and protocols for both measuring receptor-ligand interactions and cell-based assays. 

One application example on the microfluidic LSI platform is the extraction of nucleic acids (NA) from a small amount of cells [126, 127] for cell-based assays. For the extraction of NA from a cell suspension, the cell membrane has to be destroyed first (chemical lysis of the cell). Afterwards, the NA are specifically separated from the residual cell components using a solid phase extraction method based on an NA affinity column (paramagnetic beads). This extraction protocol is completely implemented on the microfiuidic... 

The assays based on metabolic activity using the vital dyes are simple, sensitive and, in most cases, provide an accurate measure of the number of live cells. In recent years the metabolic activity methods have been improved with introduction of the commercial reagents and kits, which were used to monitor cell viability and proliferation in simple add-mix-measure assay protocols. These simple assay formats allow for a wider cell-based assay application and automation. However, one should exercise caution when developing any assay based on metabolic activity of cells because there are several drawbacks with these types of... 

Fig. 2 Increasing content recovery by coupling luciferase-based assays with high-throughput biochemical readouts. The same cell line subjected to the luciferase-based assay protocol (HCT116 cells) is evaluated here for its reliability In reporting a biochemical readout (p53 expression) by dot blot analysis. Cells transfected with indicated expression construct and pathway reporters in a 96-well culture plates were lysed 48 h posttransfection. Following luciferase activity measurements (data not shown), protein from spent lysates was immobilized on nitrocellulose using a liquid handler and filtration manifold, (a) p53 and p-actin protein levels detected using protein-specific antibodies, infrared fluorescent dye-coupled secondary antibodies (with emissions at 680 and 800 nM), and the Li-COR imaging system. Columns of lysate corresponding to cells transfected with p53 DNA are boxed, (b) Quantification of the p53 to p-actin protein ratio...

The most recently developed model combined the rapid and simple 1-step protocol for the differentiation of hiPSC into HPTC-like cells (section A Simple 1-Step Protocol for the Differentiation of hiPSC into HPTC-Like Cells within 8 Days ) with the IL6/IL8-based assay and data analyses by machine learning (Kandasamy et al., 2015) (Fig. 23.3 and Table 23.1). Thirty compounds were screened. As no harvesting of the cells was required due to the high purity of HPTC-like cells derived with this protocol, the same plate in which the cells were differentiated could be used for compound screening on the evening of day 8 (Fig. 23.3). On the next morning the lysates could be harvested for qPCR analysis. Analysis of the results was performed with the RF classifier and 10-fold... 

Several established protocols have been adapted for 96-well plate readers including catalase, hyaluronidase, acetylcholinesterase, protein phosphatases and membrane-bound ATPases (22-26). In several instances these have involved novel protocols that are well suited to the ELISA format. For example, a sensitive, rapid microtitre-based assay for hyaluronidase activity was described by Frost and Stem (23). The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtitre plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtitre plate, artefacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples, with an interassay variation of less than 5%. 

While many of the specific steps are unique to the assay described in the protocol, certain general principles apply to many cell-based phenotypic assay screens. 

Two main protocols have been devised for carrying out mutation assays with mouse lymphoma L5178Y cells, plating the cells in soft agar or a fluctuation test approach. The latter is described in the following section, based on Cole et al. (1986). The reader is referred to Clive et al. (1987) for a full description of the soft-agar method. 

---