Cellular techniques
- Overview
- Assessment methods
- Learning objectives
- Contents
- Full programme
- Delivery method
- Teaching methods
- Contacts/Info
No presequisites are needed.
The exams will be in the number planned by the Degree Course. Learning is verified by submitting a written report on one of the laboratory experiences and a written exam on the basic principles of the laboratory methods studied and the discussion of the results obtained.
During the exam, the critical analysis skills and independent judgment of the students on the main topics of the course will be tested. Furthermore, knowledge of the topics covered in class and during the exercises, summarized in the didactic material available on the e-learning site, will be asked.
The exam will be evaluated based on the answers to the questions asked and on the correct use of scientific terminology in the presentation of the arguments; the behavior and participation of the student during the laboratory lessons will be also take in consideration. The final mark, expressed out of thirty, will be determined by the sum of the score obtained for the report presented (up to 3 points) and the mark of the written exam (up to 28).
The aim of the course is to provide the essential foundations of the most common laboratory techniques used in the biomedical field, with particular attention to methods of pharmacology and cellular and molecular toxicology. The student will be provided with theoretical information, together with practical and applicative details, at the basis of these methods. Overall, the student will be able to understand cellular and molecular processes considering biochemical, physiological and molecular biology aspects.
This knowledge completes the training of a graduate in Biological Sciences with solid and up-to-date skills in the fundamental sectors of life sciences, also based on the use of the methods acquired both in the biomolecular, health, and biotechnological fields, without excluding the environmental one.
Specifically, at the end of the course, the student will be able to:
1. Adequately describe the basic biomedical experimental methods.
2. Work independently in a laboratory.
3. Evaluate the experimental procedures performed.
4. Scientifically discuss the evaluation methods used and the experimental data obtained, applying, when necessary, mathematical and biostatistic knowledge.
5. Choose the right technique for a certain purpose.
6. Use cell cultures as an experimental model.
7. Integrate the acquired knowledge in a multidisciplinary context.
• Cell cultures and methods for the evaluation of cell viability: introduction to the use of cell cultures as an experimental model, advantages and disadvantages, initiation of a cell culture, work in asepsis, characteristics of the external environment (substrate, gas phase, pH and temperature, media and supplements), methods of growth and maintenance of a cell culture (medium replacement, subculture, growth curve), storage, contamination (bacteria, yeast, mycoplasma, cross-contaminations), three-dimensional cultures (characteristics and techniques to obtain them). Main methods for the evaluation of viability in cellular systems (permeability, functional, morphological and reproductive tests).
• Cell death mechanisms: types of cell death (apoptosis, necrosis, senescence, mitotic castastrophe, autophagy), their biochemical and morphological characteristics. Methods for evaluating the different types of death (electron microscopy, Tunel assay, comet assay, annexin staining, DNA fragmentation, acridine orange staining, nuclei staining, -galactosidase assay).
• Flow cytometry: basic principles, advantages and disadvantages, applications, scheme of a flow cytometer (fluidic system, optical system, electronic system and software for data analysis), fluorescence, instrument setting, data representation. Examples of use (cytogram, evaluation of cytoplasmic condensation and membrane permeability, evaluation of apoptosis, cell cycle analysis).
• Western blot analysis: basic principles, advantages and disadvantages, electrophoretic separation (polyacrylamide gel, SDS-page), transfer of the protein pattern on the membrane (by diffusion, capillarity or convection blotting, electrophoretic blotting), visualization of the proteins of interest (general and specific).
• DNA damage study: cytogenetic studies and micronucleus tests (basic principles, criteria for the selection of cells to be observed, examples).
• Molecular biology methods: mitochondrial DNA extraction, PCR
• Methods of transfection: chemical and physical methods and use of viruses. Advantages and disadvantages of the methods
• Cell cultures and methods for the evaluation of cell viability: introduction to the use of cell cultures as an experimental model, advantages and disadvantages, initiation of a cell culture, work in asepsis, characteristics of the external environment (substrate, gas phase, pH and temperature, media and supplements), methods of growth and maintenance of a cell culture (medium replacement, subculture, growth curve), storage, contamination (bacteria, yeast, mycoplasma, cross-contaminations), three-dimensional cultures (characteristics and techniques to obtain them). Main methods for the evaluation of viability in cellular systems (permeability, functional, morphological and reproductive tests).
• Cell death mechanisms: types of cell death (apoptosis, necrosis, senescence, mitotic castastrophe, autophagy), their biochemical and morphological characteristics. Methods for evaluating the different types of death (electron microscopy, Tunel assay, comet assay, annexin staining, DNA fragmentation, acridine orange staining, nuclei staining, -galactosidase assay).
• Flow cytometry: basic principles, advantages and disadvantages, applications, scheme of a flow cytometer (fluidic system, optical system, electronic system and software for data analysis), fluorescence, instrument setting, data representation. Examples of use (cytogram, evaluation of cytoplasmic condensation and membrane permeability, evaluation of apoptosis, cell cycle analysis).
• Western blot analysis: basic principles, advantages and disadvantages, electrophoretic separation (polyacrylamide gel, SDS-page), transfer of the protein pattern on the membrane (by diffusion, capillarity or convection blotting, electrophoretic blotting), visualization of the proteins of interest (general and specific).
• DNA damage study: cytogenetic studies and micronucleus tests (basic principles, criteria for the selection of cells to be observed, examples).
• Molecular biology methods: mitochondrial DNA extraction, PCR
• Methods of transfection: chemical and physical methods and use of viruses. Advantages and disadvantages of the methods
The teaching activities include lectures, classroom exercises and laboratory experiences.
• Lectures will be carried out with the help of slides.
• Classroom exercises involve the use of slides and the active participation of students with the aim of carrying out, first under the guidance of the teacher and then independently, problems related to laboratory experiences. In particular, dilution calculations and / or theoretical and practical problems concerning the experimental program will be carried out.
• Practical activities in the laboratory will be introduced during the lectures. The experiences will allow to acquire the basic pharmacology and cellular and molecular toxicology techniques. The laboratory lessons are held at the Molini Marzoli laboratories, via A. da Giussano 10, Busto Arsizio. To each student will be assigned a workstation equipped with all the necessary equipment (pipettors, tips, test tubes, reagents, etc.) and, for each lesson, the protocol to be followed will be provided. During the laboratory module, continuous assistance in the classroom will be ensured by the teacher and one or more assistant.
The chair of the course is available for meetings with students, following appointment via e-mail (marzia.gariboldi@uninsubria.it).