Evaluation of Cellular Processes by In Vitro Assays

In order to get the intricate biology of living organisms, the researchers started using the basic unit of life i.e. “Cell” for elucidating the intricate mechanisms. This led to the basis of cell culture studies, where cells grown in controlled artificial conditions simulate the conditions prevailing in natural ones, therefore, presumed to act as those in in vivo conditions. The introduction of cell culture techniques has helped a lot in understanding the physiological processes like cell signalling, neurobiology, cell proliferation, pathogenesis of diseases, apoptosis and even more. In the following chapter, we will discuss the basics of cell culture including the equipments, chemicals and different types of materials required for cell culture. The techniques used for maintenance, preservation and authentication of cell lines are also included.

In order to evaluate the regulation, expression or activity of a gene, it is necessary to transfer the gene or its manipulated form into the in vitro systems. Since the mammalian cells do not uptake the foreign DNA efficiently, the availability of effective methods for introducing genes into the cells is essential. The transfection methodology has developed rapidly and diversely. Each year many new products and technologies are launched with improved efficiency and less cytotoxicity. In the following chapter, we will discuss the advantages and disadvantages of different methods of transfection.

Apoptosis is a critical process having widespread biological significance; regulating the differentiation, proliferation, maintenance and sculpturing organs and tissues, functioning of immune system and the elimination of defective harmful cells. The word apoptosis is derived from Greek word meaning “απόπτωσις” meaning “falling off of petals from flowers”. It is actually programmed cell death essential for normal metabolism. In this chapter, we will provide general overview of various technical approaches for detecting apoptotic cells. The features, advantages and disadvantages of different methods are also discussed.

Cell viability and cytotoxicity assays are used usually for screening of drugs and cytotoxicity tests of chemicals. Cell viability, cell proliferation and many important live-cell functions can be stimulated or monitored with various chemical and biological reagents. The present chapter is aimed to discuss the various available method

The reactive oxygen species produced endogenously are essential to life, being involved in several biological functions. However, when produced at higher levels, these reactive species become highly harmful, causing oxidative stress through the oxidation of biomolecules, leading to cellular damage. The study in the field of ROS associated biological functions and/or deleterious effects requires new sensitive and specific tools in order to enable a deeper insight on its action mechanisms. Here, we discuss several methods related to the detection of reactive oxygen species.

Protein-protein interactions are central to every cellular process like DNA replication, transcription, translation, splicing, signal transduction and cell cycle control. To elucidate the function of a gene, we need to determine the function of the gene’s encoded protein product. Identifying the role of a protein needs understanding about the proteins that interact with each other for functioning of a particular biological pathway. Assessment about function of a protein can be made by understanding protein-protein interaction studies. These inferences are based on the fact that the role of unknown proteins may be elucidated if captured through their interaction with a protein target of known function. Thus in this chapter, we attempt to summarize different in vitro methods used to identify proteins- protein interactions and to assess the strengths of these interactions.

The biological network is intricately woven by several entities which work in cellular system in a co-ordinated manner as a result of different types of interactions (protein-protein interactions, protein-RNA and protein-DNA interactions). Among them, the protein-nucleic interactions play an important role in regulating cell function and disruption among these interactions leads to catastrophic consequences within the biological system. The protein-nucleic acid interactions are integrated into several key cellular processes which include regulation of gene expression, replication, recombination, repair, translation, transcription, packaging of nucleic acids and the formation of cellular machinery. In this chapter, we will discuss the several methods by which we can analyse protein-nucleic acid interactions.