Advanced Cell and Molecular Techniques Protocols for In Vitro and In Vivo Studies

This detailed volume explores laboratory protocols and methodologies for studying cell cultures, genetic analysis, and various biochemical assays. The chapters within this book cover a wide range of techniques, from basic cell culture methods to more specialized assays used in the study of genotoxicity, apoptosis, and gene expression, not only relevant to academic researchers but also to professionals in the pharmaceutical, biotechnological, and clinical fields. Written for the Springer Protocols Handbooks series, each protocol has been carefully compiled to provide step-by-step instructions, ensuring clarity and reproducibility.

Authoritative and practical, Advanced Cell and Molecular Techniques: Protocols for In Vitro and In Vivo Studies serves as both a hands-on guide and a source of inspiration for future research as the fields of molecular biology and toxicology continue to evolve.

Cell Culture Techniques.- Solutions Preparation.- Standard Operating Procedure (SOP) for Maintaining Sterility in the Laminar Flow Hood During Cell Culture Procedures.- Protocol for Maintenance, Isolation, and Primary Culture of Ehrlich Tumor Cells.- Stem Cell Cultivation and Maintenance.- Protocol for Isolation of Stem Cells from Rabbit Adipose Tissue.- Protocol for Isolation of Lung Fibroblasts from Mouse Tissue.- Protocol for Isolation of Mice Fat Stem Cells.- Protocol for Isolation of Mouse Adipose-Derived Stem Cells (ADSCs).- Protocol for Isolation of Bone Marrow Stem Cells.- Extraction of Mesenchymal Stem Cells (MSCs) from Human Liposuction Fat.- Human Bone Marrow Extraction Protocol.- Mesenchymal Stem Cells (MSCs) Differentiation Protocols.- cDNA Synthesis and Quantitative PCR (qPCR) for Gene Expression Analysis.- Preparation and Optimization of Reagents for Genetic Analysis and Infectious Disease Diagnosis.- DNA and RNA Extraction from Plasma Using Magnetized Silica.- RNA Extraction Using Trizol (6-Well Plate Method).- Agarose Gel Electrophoresis for RNA Integrity Assessment.- DNA Purification Using the Wizard Plus SV Minipreps DNA Purification System (Promega).- Detection of Reactive Oxygen Species (ROS) in Embryos.- Determination of Protein Concentration: Sedmak and Grossberg Method.- Cell Sorting of Fluorescent Zebrafish Cells.- Cell Disaggregation for Flow Cytometry Analysis of Fluorescent Zebrafish Cells.- RNA Extraction from Zebrafish Embryos for RT-qPCR.- Extraction of Genomic DNA.- RNA Extraction from HEK293.- Agarose Gel Preparation for RNA and DNA Electrophoresis.- Zebrafish Genotyping by Fin Clipping.- In Situ Hybridization of Zebrafish Embryos.- Concepts and Practices in Flow Cytometry.- In Situ Apoptosis Assay.- Flow Cytometry Protocols for Assessing Membrane Integrity, Cell Cycle, and Apoptosis in Treated Cells.- Leukocyte Reading Protocol on the Flow Cytometer.- Flow Cytometry Protocol for Mouse Sperm Cells (BD Accuri C6+).- Extraction of Yolk Proteins from 24 HPF Zebrafish Embryos.- PFA Fixation for Zebrafish Embryos.- Thioflavin T (ThT) Fluorescence Measurement.- Alcian Blue Staining Protocol.- Zebrafish Embryo Staining with Nicotinamide Adenine (NA).- Western Blot on Zebrafish Embryos.- In Vitro Micronucleus Assay in Binucleated CHO-K1 Cells.- In Vitro Assessment of Genotoxicity Using the Comet Assay in CHO-K1 Cells.- Treatment and Cell Collection for Chromosomal Aberration Test.- Electrophoretic Mobility Shift Assay (EMSA): Non-Radioactive, Band Shift, Gel Shift, or Gel Retardation.- MTT Assay Protocol.- Spectropolarimeter (Jasco-1500) Operation Protocol.- Immunoprecipitation Protocol (Tuning).- qPCR EvaGreen Hot FIREPol Mix.- Synthesis of RNA-Digoxigenin Probe.- Thermal Difference Spectra (TDS).- Generation of Competent Bacteria.- Retro Transcription via STEM LOOP.- In Vitro Transcription with the mMessage mMachine SP6 Transcription Kit.- Transfection in HEK293 Cells.- HeLa Transfections with Lipofectamine 3000.- Chemical Competent Cell Transformation.