1st Edition
Advanced Mammalian Cell Culture Techniques Principles and Practices
This up-to-date book compiles both basic and advanced laboratory techniques of mammalian cell culture. It is divided into four major sections encompassing the basics of cell culture, nucleic acid and protein isolation, cell-staining techniques, and cell transfection and single-cell analysis. The topics include aseptic handling, media preparation, and passaging of cells. The book also outlines downstream assays such as nucleic acid and protein isolation from in-vitro cell cultures.
Key Features:
- Covers cellular staining using fluorescent dyes, genetic manipulation of cells via transfection, and an introduction to single-cell analyses
- Discusses basics in cell culture and downstream applications including gene and protein expression analysis
- Includes the principles underlying each of the techniques and provides a detailed methodology to practice
- Explores the whole range of techniques – from basic to downstream applications and advanced methods
The book is essential for students and researchers in the field of life sciences, biotechnology, genetics, and molecular biology.
Preface
Acknowledgments
Editor Biographies
Contributors
Foreword A and B
SECTION 1. BASICS IN CELL CULTURE
1. Highlights: History of Cell Culture and Its Applications
Goutham V. Ganesh, Karan Naresh Amin, Sundhar Mohandas, R. Senthilkumar, and K.M. Ramkumar
2. Aseptic Technique and Essentials in Cell Culture
Goutham V. Ganesh, Milan K.L., Sundhar Mohandas, R. Senthilkumar, and K.M. Ramkumar
3. Laboratory Safety
Goutham V. Ganesh, Kannan Hairthpriya, Dhamodharan Umapathy, Md Enamul Hoque, and K.M. Ramkumar
4. Preparation of Media for Cell Culture
Milan K.L., Goutham V. Ganesh, Sundhar Mohandas, Md Enamul Hoque, and K.M. Ramkumar
5. Primary Cell Culture
Milan K.L., Goutham V. Ganesh, Dhamodharan Umapathy, Md Enamul Hoque, and K.M. Ramkumar
6. Passaging of Cell Lines
Milan K.L., Goutham V. Ganesh, Dhamodharan Umapathy, Md Enamul Hoque, and K.M. Ramkumar
7. Cell or Tissue Culture Models and Cell Line Immortalization
Goutham V. Ganesh, Milan K.L., Dhamodharan Umapathy, and K.M. Ramkumar
8. Counting of Cells by Hemocytometer
Milan K.L., Goutham V. Ganesh, Sundhar Mohandas, and K.M. Ramkumar
9. Cryopreservation of Cells
Milan K.L., Karan Naresh Amin, Sundhar Mohandas, Md Enamul Hoque, and K.M. Ramkumar
10. Thawing of Cells
Milan K.L., Goutham V. Ganesh, Sundhar Mohandas, and K.M. Ramkumar
11. Detection of Cacterial and Fungal Contamination
Milan K.L., Karan Naresh Amin, Dhamodharan Umapathy, and K.M. Ramkumar
12. Testing for Mycoplasma Contamination
Milan K.L., Ravichandran Jayasuriya, Dhamodharan Umapathy, and K.M. Ramkumar
13. Cellular Cytotoxicity
Milan K.L., Kannan Harithpriya, Dhamodharan Umapathy, Md Enamul Hoque, and K.M. Ramkumar
SECTION 2. ISOLATION AND ANALYSIS OF NUCLEIC ACIDS AND PROTEINS
14. Isolation of Total RNA and Assessment of Purity and Concentration
Milan K.L., Kannan Harithpriya, Dhamodharan Umapathy, R. Senthilkumar, and K.M. Ramkumar
15. Target Gene Amplification and Expression by qRT-PCR
Milan K.L., Kannan Harithpriya, Dhamodharan Umapathy, R. Senthilkumar, and K.M. Ramkumar
16. Cell Lysis, Protein Extraction Using RIPA Buffer
Kannan Harithpriya, Ravichandran Jayasuriya, Dhamodharan Umapathy, and K.M. Ramkumar
17. Estimation of Protein by Bradford’s Assay
Kannan Harithpriya, Ravichandran Jayasuriya, Dhamodharan Umapathy, and K.M. Ramkumar
18. SDS-PAGE Analysis
Kannan Harithpriya, Goutham V. Ganesh, Dhamodharan Umapathy, and K.M. Ramkumar
19. Immunoblotting
Kannan Harithpriya, Karan Naresh Amin, Dhamodharan Umapathy, and K.M. Ramkumar
SECTION 3. CELL STANING TECHNIQUES
20. Cell Nuclear Staining by DAPI
Ravichandran Jayasuriya, Karan Naresh Amin, Sundhar Mohandas, R. Senthilkumar, and K.M. Ramkumar
21. Immunofluorescence Staining
Ravichandran Jayasuriya, Kannan Harithpriya, Sundhar Mohandas, R. Senthilkumar, and K.M. Ramkumar
22. Estimation of Intracellular ROS using H2DCFDA Molecular Probes
Ravichandran Jayasuriya, Karan Naresh Amin, Sundhar Mohandas, and K.M. Ramkumar
23. Analysis of Cell Cycle Regulation and Apoptosis Using Annexin-V and Propidium Iodide (PI) Staining
Ravichandran Jayasuriya, Karan Naresh Amin, Sundhar Mohandas, and K.M. Ramkumar
24. Estimation of Chemokines and Cytokines Using Bio-Plex Assay
Ravichandran Jayasuriya, Karan Naresh Amin, Sundhar Mohandas, and K.M. Ramkumar
SECTION 4. CELL TRANSFECTION AND SINGLE-CELL ANALYSIS
25. Reporter Gene Construction and Assay
Karan Naresh Amin, Ravichandran Jayasuriya, R. Senthilkumar, and K.M. Ramkumar
26. Vector Transfection and Validation
Karan Naresh Amin, Ravichandran Jayasuriya, R. Senthilkumar, and K.M. Ramkumar
27. Single-Cell Analysis
Karan Naresh Amin, Ravichandran Jayasuriya, R. Senthilkumar, Md Enamul Hoque, and K.M. Ramkumar
Index
Biography
Dr Md Enamul Hoque is a Professor at the Department of Biomedical Engineering in the Military Institute of Science and Technology (MIST), Dhaka, Bangladesh. Before joining MIST, he served in several leading positions in some other global universities such as Head of the Department of Biomedical Engineering at King Faisal University (KFU), Saudi Arabia; Founding Head of Bioengineering Division, University of Nottingham Malaysia Campus (UNMC) and so on. He received his PhD from the National University of Singapore (NUS), Singapore in 2007 with a globally prestigious scholarship from the Singapore Government. He also obtained his PGCHE (Post Graduate Certificate in Higher Education) from the University of Nottingham, UK in 2015. He is a Chartered Engineer (CEng) certified by the Engineering Council, UK; Fellow of the Institute of Mechanical Engineering (FIMechE), UK; Fellow of Higher Education Academy (FHEA), UK and Member, World Academy of Science, Engineering and Technology. To date, he has published around 108 Journal Papers, 5 Special Issues in Journal, 11 books, 70 book chapters, and 100 international conference presentations/proceedings. Besides, 56 manuscripts (19 Journal Papers + 7 Books + 28 Chapters + 2 Conference Proceedings/Presentations) are under review. His publications have attracted about 3305 citations with 26 h-index and 67 i-10 Index (https://scholar.google.com/citations?user=aRvzzrEAAAAJ&hl=en) as per Google Scholar citationreport. He received the Outstanding Nano-scientist Award in the International Workshop on Recent Advances in Nanotechnology and Applications (RANA-2018) held on 7 - 8 September 2018, AMET University, Chennai, India. His major areas of research interests include (but are not limited to) Bioceramics, Biomaterials, Biocomposites, Biopolymers, Nanomaterials, Biomedical Implants, Rehabilitation Engineering, Rapid Prototyping Technology, Stem Cells and Tissue Engineering. As per AD Scientific Index, he has World Scientist Ranking (as on 21Dec 2022) in Biomedical Engineering as 1st (Best) in the country (Bangladesh), 328 in Asia, and 1536 in the World; https://www.adscientificindex.com/scientist/md-enamul-hoque/424943.
Dr. Senthilkumar Rajagopal is an Associate Professor/Ramalingaswami Re-entry fellow [DBT] of Department of Biotechnology, REVA University, Bangalore, INDIA. He secured PhD in Biochemistry from Annamalai University, INDIA. He did his three Post-doctoral researches in Anesthesiology in University of Virginia, Charlottesville, Virginia, followed by Department of Cancer Immunology & AIDS, Dana Farber Cancer Institute, Harvard University, Boston, Massachusetts and, Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia, USA. Dr. Senthilkumar is a recipient of Rameshwardasji Birla Smarak Kosh Endowment award from Mumbai Medical Trust, Mumbai, INDIA and Ramalingaswami Re-entry Fellowship from Department of Biotechnology, Ministry of Science and Technology, Government of India. He has more than 18 years of research experiences in various fields such as Alcoholic hepatotoxicity, Voltage gated calcium channels, protein kinase C phosphorylation, ADAM family, Smooth muscle physiology, G-protein coupled receptor signaling pathways, Neurotransmitters transporters to Central Nervous system. He has published nearly 75 research articles in various peer-reviewed international journals, 35 book chapters, 10 abstracts, 65 conferences proceedings/abstracts and attended various workshops/seminars in India, Canada and USA. He has reviewed and screened the abstracts for National Biotechnology Conferences and Annual Biomedical Research Conference for Minority Students (ABRCMS) and also served the special judge for Biophysical Society award at Virginia Piedmont Regional Science Fair. He conducts research in the area of G-protein coupled receptor mediated intracellular mechanisms by Alcohol-induced digestive disorders in the gastrointestinal tract. Currently, he is mentoring graduate students and having research projects from various funding agencies.
Dr. Ramkumar is a Professor of Biotechnology at SRM Institute of Science and Technology, India. He received his doctoral degree from Bharathidasan University, Tiruchirapalli, India, where he was trained in cell signaling networks, particularly pancreatic β-cell apoptosis. He expanded his realm into the cellular pathophysiology of diabetes during his post-doctoral fellowship at the Chonbuk National University Medical School, Republic of Korea. Further, during his research at the Stanford University School of Medicine, USA, he developed molecular imaging methods to study cellular and molecular pathways in vitro and in vivo.
During his academic tenure at SRMIST, Dr. Ramkumar established Cell Culture Laboratory with a 'State of Art' infrastructure. He has made several contributions to the cellular signaling network in diabetes. He has developed a high throughput cell-based assay for screening signaling molecules that restore cellular homeostasis. His research interests span the identification of treatment targets for the management of diabetes. Much of his work has been on understanding the transcription factors involved in redox signaling associated with diabetes and its complications. He has also investigated the epigenetic regulation of cellular mediators involved in the pathophysiology of various diseases. Dr. Ramkumar has conducted several "Hands-on Training Workshops" related to Cell culture.
He has over 150 scholarly contributions, including 100 peer-reviewed research papers, and based on his research accomplishments, Dr. Ramkumar has been ranked among the top 2% scientists consecutively for two years, as per the global list released by Stanford University, USA. His research has received more the 3 Crores of funding from several Government agencies, including the Science and Engineering Research Board (SERB), Indian Council of Medical Research (ICMR), Department of Biotechnology (DBT), and Department of Science and Technology (DST) and Industries. He is also a recipient of various awards from Government agencies including “Young Scientist Award” from DST and “SERB International Research Experience (SIRE)” award from SERB.
Dr. Ramkumar has established Research Cohort with several hospitals and closely associates with clinicians, translating research from bench to bedside.
