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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
This handbook presents information on different cell culture assays which can be used to perform experimental analysis. Readers are introduced to the basics of in vitro cell cultures followed by a comparative analysis of different experimental protocols d
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Seitenzahl: 154
Veröffentlichungsjahr: 2018
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Evaluation of Cellular Processes
by In Vitro Assays
BENTHAM SCIENCE PUBLISHERS LTD.
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FOREWORD
This book provides hands on information about the various in vitro techniques and is preceded by the information about the principles and the basics of the technique. The authors have clearly and concisely discussed several concepts and methods which will be useful for the under-graduate and post-graduate students during their laboratory courses. The manuscript will equally contribute to the day-to-day research work of the scholars.
I strongly recommend publishing of this book and I wish best of luck to all the authors.
This book provides hands on information about the various in vitro techniques and is preceded by the information about the principle and the basics of the technique. The authors have clearly and concisely discussed several concepts and methods which will be useful to the under-graduate and post-graduate students during their laboratory courses. The manuscript will equally contribute to the day-to-day research work of the scholars.
I strongly recommend publishing of this book and I wish best of luck to all the authors.
PREFACE
During the course of my doctoral degree, I was very much interested in the methods involved in the understanding effect of drugs in in vitro conditions. The students and scholars often find it confusing to select a particular assay on cell viability, cell proliferation and apoptosis. While teaching at the post-graduate level, I got to know about the problems our students face in understanding the basics of in vitro assays. Keeping in view these facts, I tried to address these concerns by providing a basic introduction about various assays in the form of this book. The book gives comparative analysis of different assays and discusses the advantages and disadvantages related to it. Each experimental protocol is preceded by the information about the principle and the basics of the technique.
I hope my little effort will benefit the students and scholars who are interested to know the basic fundamentals of cellular assays. To make this book better, the criticism and suggestion are most welcomed.
ABBREVIATIONS
Taseen Gul,Henah M Balkhi,Ehtishamul HaqAbbreviations
BACBacterial Artificial ChromosomeBpBase pairBrdU5-bromo-2’-deoxyuridinecDNAComplementary DNACFSECarboxy fluorescein diacetate succinimidyl esterChIPChromatin ImmunoprecipitationDAPI4,6-Diamidino- 2-phenylindoleDIGDigoxigeninDMEMDulbecco’s Minimal Essential MediaDMSODimethyl sulphoxideDNTB5-5'-Dithiobis [2-nitrobenzoic acid]ELISAEnzyme Linked Immunosorbent AssayEMSAElectrophoretic Mobility Shift AssayFITCFluorescein IsothiocyanateFBSFetal Bovine SerumHRPHorseradish peroxidiseICInhibitory ConcentrationILInterleukinJC-15,5',6,6'-tetrachloro- 1,1',3,3' tetraethylbenzimidazolcarbocyanine iodideKDaKilo DaltonLDHLactate DehydrogenasemRNAmessenger RNAmiRNAmicro RNAMDAMalondialdehydeNAD+Nicotinamide adenine dinucleotideNO3-NitrateNO2-NitriteNONitric oxidePAGEPolyacrylamide Gel ElectrophoresisPARPPoly ADP-ribose polymerasePIPropidium iodideRBRetinoblastomaROSReactive oxygen speciesRT-PCRReal time polymerase chain reactionT-DNATransfer DNATNF-αTumor necrosis factor alpha.TNFR1Tumor necrosis factor receptor 1TUNELTerminal deoxynucleotidyl transferase dUTP nick end labellingTBAThiobarbituric aciduMmicromolarWST{2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium}XTT2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.List of Contributors
Basics of In Vitro Cell Culture
Taseen Gul,Henah M Balkhi,Ehtishamul HaqAbstract
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.
INTRODUCTION
Characteristic feature of all animals is that they are multi-cellular, consisting of many different cells, with specialized functions. Basic metabolic pathways in different cells are same with similar organelles however each cell also has a specific function and thus have unique expression of some of these components to perform specific function within the organism. Depending on specific roles, each cell type has a specific gene and protein expression with a characteristic shape, size, structure and function. They are said to have differentiated and are highly organised structures with specialised functions. The science and technological interventions have done marvels in understanding the cellular phenomenon and allowed us to grow cells even under artificial conditions. Isolated cells, tissues or organs can be grown in plastic dishes when they are kept at defined temperatures using an incubator and supplemented with a medium containing cell nutrients and growth factors.
First detailed study on in vitro culture of cells was made by Jolly in 1903. He studied cell survival and cell division in leukocytes of a salamander. Earlier
during such studies, experiments were conducted on chunks of tissues and as such the name 'tissue culture' was used. However now, the term tissue culture is used for in vitro culture of cells only when cell culture is kept for more than 24 hours. In the evolution of animal cell culture studies, the major breakthrough was done by Ross Harrison, whose fundamental work in animal cell culture is considered as a corner stone of animal cell culture in science [1]. In 1907 he experimented on clotted lymph fluid using explants of frog embryo and observed cell proliferation in a depression slide. This technique continued to be used since then. It involves suspension of a drop over a depression in a microscopic slide sealed by a cover slip. This technique was further developed by Burrows with the use of plasma clots. Although they could evolve the technique significantly but the major hassle in the cell culture was the maintenance of the cultures without contamination from bacterial or other microbial cells. Since bacterial cells grew at a very fast rate in comparison to growth rates of animal cells, even a low-level contamination lead to unacceptable rate of contamination. To overcome this problem a surgeon Alexis Carrel, introduced strict aseptic techniques for cell culture in vitro. In order to sustain aseptic conditions for cell culture he introduced the 'Carrel flask’ for aseptic subculture of cells and thus became the primogenitor of modern tissue culture. However, it was a lengthy procedure and difficult to repeat creating hurdles to adopt cell culture as a routine laboratory technique. In 1912, the first cell culture for a long period of 34 years was successfully carried out by Carrel, who started to culture chick embryo heart cells. This led to the imprecise belief that cells could be cultured for an indefinite period if given the appropriate conditions. Later it was observed that cell growth was maintained by the use of embryo extracts, but new cells were being continuously added to the culture during medium replenishment. In 1961, Hayflick and Moorhead established the finite capacity for cell growth. This was followed by a significant advancement in culturing technique by introduction of Trypsinization, by Rous and Jones in 1916. Trypsinisation involved the treatment of cells by the proteolytic enzyme trypsin to free cells from tissue matrix. Tissue culture contains a mixture of cell types and to produce homogeneous cell strains from such tissue cultures trypsinization was a breakthrough marking the start of animal cell culture techniques. Table 1.1 represents the major events that took place in the history of advancements in the field of culture techniques [2].
1.1. Tissue Culture and Its Types
In vitro culture of cells, tissues and organs is commonly referred as tissue culture. Tissue culture is cultivation of animal as well as plant cells in vitro. Tissue culture is classified into three major groups; organ culture, explant culture, and cell culture [3].
Organ Culture: The three-dimensional culture of tissues so that some or all of the histological features of the tissues are retained. The organ culture is carried out in such a way that cells under cultivation differentiate in a proper architecture. Organs cultured are able to accurately function in various states and conditions as actual in vitro organ itself. Organ culture could be carried out through different methods as:
Plasma Clot Method:
