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Full Description
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.
Contents
SECTION 1. BASICS IN CELL CULTURE, 1. Highlights: History of Cell Culture and Its Applications, 2. Aseptic Technique and Essentials in Cell Culture, 3. Laboratory Safety, 4. Preparation of Media for Cell Culture, 5. Primary Cell Culture, 6. Passaging of Cell Lines, 7. Cell or Tissue Culture Models and Cell Line Immortalization, 8. Counting of Cells by Hemocytometer, 9. Cryopreservation of Cells, 10. Thawing of Cells, 11. Detection of Bacterial and Fungal Contamination, 12. Testing for Mycoplasma Contamination, 13. Cellular Cytotoxicity, SECTION 2. ISOLATION AND ANALYSIS OF NUCLEIC ACIDS AND PROTEINS, 14. Isolation of Total RNA and Assessment of Purity and Concentration, 15. Target Gene Amplification and Expression by qRT-PCR, 16. Cell Lysis, Protein Extraction Using RIPA Buffer, 17. Estimation of Protein by Bradford's Assay, 18. SDS-PAGE Analysis, 19. Immunoblotting, SECTION 3. CELL STANING TECHNIQUES, 20. Cell Nuclear Staining by DAPI, 21. Immunofluorescence Staining, 22. Estimation of Intracellular ROS using H2DCFDA Molecular Probes, 23. Analysis of Cell Cycle Regulation and Apoptosis Using Annexin-V and Propidium Iodide (PI) Staining, 24. Estimation of Chemokines and Cytokines Using Bio-Plex Assay, SECTION 4. CELL TRANSFECTION AND SINGLE-CELL ANALYSIS, 25. Reporter Gene Construction and Assay, 26. Vector Transfection and Validation, 27. Single-Cell Analysis
