cellular techniques in biomedical research
- Overview
- Assessment methods
- Learning objectives
- Contents
- Bibliography
- Delivery method
- Teaching methods
No presequisites are needed.
The final assessement included a write relation about one of the laboratory experiments and a written exam.
Students will acquire theoretical and practical knowledge about the main techniques used in a cellular and molecular pharmacology and toxicology laboratory (for example: use of cell culture, methods used to evaluate cell viability and apoptotic or autophagic cell death, biochemical and molecular biology methods etc..).
Students will acquire independent management of practical experiments and ability in understanding a correct protocol design and ability in presenting and discussing methods and obtained results.
THEORETICAL LESSONS (2 CFU, 16h)
- Cell culture: introduction to the use of cell culture, advantages and disadvantages, initiating a culture, design and layout of the laboratory, aseptic techniques, the culture environment (substrate, gas phase, pH and temperature, media and supplements), maintenance of the culture (replacement of medium, subculture, growth cycle), storage, contaminations (bacteria, yeast, fungi, molds, mycoplasms, cross contaminations), three-dimentional culture (characteristics and way to get it). Evaluation of cell viability (permeability, functional, morphological and reproductive tests). Results analysis.
- Cell death: morphological and biochemical characteristics of apoptosis, necrosis, senescence autophagy. Methods to evaluate cell death (electronic microscopy, tunel assay, comet assay, DNA fragmentation tests, etc). Results analysis
- Flow cytometry: principles, advantages and disadvantages, the instrument (fluidic, optical, electronic systems, software), fluorescence, setting of the instrument, data representation. Examples (cytogram, cell condensation and membrane permeability, evaluation of apoptotic cell death, cell cycle analysis). Results analysis.
- Western Blot analysis: principles, advantages and disadvantages, gel electrophoresis of proteins, transfer, detection of proteins (general or specific). Results analysis.
- DNA damage: cytogenetic studies and micronuclei test. Results analysis.
- Molecular Biology methods: mitochondrial DNA extraction, PCR. Results analysis.
- Transfection: chemical, physical and use of virus. Advantages and disadvantages of the methods described.
LABORATORY LESSONS (4 CFU, 48h)
Students will be divided into small groups and will work in laboratories with laminar flux hood.
- Subculture of suspention and monostrate growing cells, count by Burker emocytometer or electronic cell counter, maintainance of a fixed number of cells.
- Evaluation of the cytotoxic effect of a drug: clonogenic and MTT assay.
- Evaluation of apoptotic cell death
- Western blot analysis of whole cell lysates.
- Micronuclei test
Slide presentations and scientific articles on specific topics are supplied by the lecturer. For the practical part of the course, technical protocols are supplied. All material will be available on the e-learning platform.
The course includes theoretical (2CFU) and laboratory (4 CFU) lessons. During the theoretical lessons the topics will be treated with the help of slide presentations. The laboratory lessons will be conducted at the Molini Marzoli laboratories, in Busto Arsizio. Attendance at laboratory lessons is mandatory; the absence is only allowed for a lower number of hours to 25% of the total laboratory hours.