Investigation Methodologies in Animal Cell Biology and Histology

Cell cultures and their applications. Major requirement of a cell culture laboratory. Sterility assurance and contamination control. Setup, maintenance and conservation of suspension and adherent cultures: initial stage of cell culture growth, trypsinization of monolayers, evolution of cell lines, isolation of clones, cryopreservation. Types of cultures: primary cultures (organ and tissue cultures), secondary cultures, finite cell lines, continuous, massive or clonal cultures, hybridomas.

Techniques for characterizing and monitoring cells in culture. Evaluation of cell viability and proliferation (live cell labeling, colorimetric and flow cytometric assays). Evaluation of apoptosis by fluorescence microscopy and flow cytometry. Evaluation of cellular senescence.

Techniques for subcellular detection of fluorescent reporter proteins. Introduction of exogenous DNA into mammalian cells: transfection methods, transient transfections, isolation of stably-transfected cell lines. Fluorescent probes. GFP and its mutants: development and spectroscopic properties. Applications of GFP: subcellular localization of proteins by confocal microscopy, analysis of protein-protein interactions (FRET), analysis of protein dynamics (FRAP), photoactivatable GFPs.

Stem cells: origin of stem cells, characteristics of embryonic and adult stem cells. Induced pluripotent stem
cells. Culture and maintenance of stem cells and their applications in therapeutic field.

Histological techniques for the phenotypic and functional study of biological tissues: sampling, physical and
chemical fixation methods, embedding in paraffin and plastic resins, sectioning and related instruments. Staining of sections for observation under an optical microscope. Dyes: structure and chemical classification. Chromophores and auxochromes. Histomorphological, cytochemical and immunohistochemical staining.