OVERVIEW

Flow cytometry is a very flexible platform for the study and separation of cells and particles in suspension, simultaneously measuring physical parameters and fluorescence characteristics, thus solving complex biological samples. Due to its versatility, cytometry is widely applied to the study and knowledge in many fields of biology and medicine, therefore very widespread also in the laboratories of pharmaceutical companies.

AIMS AND CONTENT

LEARNING OUTCOMES

The course aims to provide the basic knowledge of a widely spread technique. In particular, the achievement of the following objectives: knowledge of the fundamental principles of flow cytometry and instrumentation; reference points and guidelines in the execution of cytometric measurements; in-depth analysis of applications for biological and medical studies; reading and interpretation of the results obtained.

AIMS AND LEARNING OUTCOMES

The course aims to provide basic knowledge of a technique widely used in laboratories not only in biomedical fields, but also in environmental and food sciences. Specifically, upon completion of the course, students must demonstrate achievement of the following objectives: understanding the fundamental principles of flow cytometry and instrumentation; understanding reference points and guidelines for performing cytometric measurements; applying appropriate cytometric protocols and standards to biological and medical studies; and analyzing and interpreting the results obtained. Detailed learning objectives and learning outcomes Attendance and participation in the training activities (lectures and laboratory work) will allow students to acquire the knowledge necessary to address the various types and approaches of cytometric measurements in the biological field.

Specifically, at the end of the course, students will demonstrate achievement of the following objectives:

- Knowledge of the fundamental principles of flow cytometry and instrumentation

- Describe the main protocols for applying cytometric measurements

- Resolve applications in the biological and biomedical fields

- Analyze the measurements performed

- Discuss the importance of the observed cytometric data

PREREQUISITES

To effectively deal with the contents of the course, the following basic knowledge is required: structure of the cell and its components, knowledge of the cell cycle, general notions of light-matter interaction and the phenomenon of fluorescence.

TEACHING METHODS

The course includes 40 hours divided between lectures delivered through multimedia presentations and presentation of cytometric files and laboratories with examples of acquisitions/analyzes and sorting.

Any Student with documented Specific Learning Disorders (SLD), or with any special needs, shall reach out to the Lecturer(s) and to the dedicated SLD Representative in the Department before class begins, in order to liase and arrange the specific teaching methods and ensure proper achievement of the learning aims and outcomes.

SYLLABUS/CONTENT

1. Cytofluorimetry: meaning/recommended bibliography, history of technology
2. Fluorescence and fluorochromes: principles and functioning, general notions about dyes for nucleic acids, for proteins
3. Fluidics: applied hydrodynamic principles
4. Optics: light, theory and materials
5. Electronics: data acquisition, analysis and representation
6. Instrumental performance procedures: instrumentation control (calibration, calibration, linearity); setting of the analysis threshold, compensation
7. Sample preparation: basic knowledge for sample preparation (fresh, fixed or frozen samples); manipulation of cell lines in culture and their preparation for cytometric investigations
(with particular reference to the adhesion crops: detachment, fixation, etc.); treatment and analysis of samples (biopsies) of solid tissues with particular reference to the various unbundling techniques; study of microvesicles.
8. Multiparametric flow cytometry:
• Preparation of the sample and preparation of the panel
• Problem solving: spill-over and compensation
• Autofluorescence, quenching, bleaching
• Setting of antibodies (staining index)
9. Management and Analysis of the data generated: Main concepts connected with the analysis of a cytometric file: notions on the different gating modes; off-line compensation of fluorescence; isotypic and isoclonic control, fluorescence minus one (FMO); Criteria for evaluating positivity; main types of representation of cytometric data.
10. The cell sorting: theory and instrumental functioning, examples of applications
11. Applications of the cytometric platform in biomedical research and diagnostics:
• evaluation of cell cycle and cell proliferation;
• evaluation of apoptosis;
necrosis and cell death;
• study of surface, intracytoplasmic and intranuclear markers;
• cell signalling;
• production of soluble factors;
• Cytotoxicity;
• Microvesicles and exosomes.
12. State of the art and new generation tools.

RECOMMENDED READING/BIBLIOGRAPHY

All the slides used during the lessons will be available on AulaWeb at the end of the series of lessons in PDF format.  Specific guidelines on the reference bibliography will be provided by the professor at the beginning of the course

TEACHERS AND EXAM BOARD

Exam Board

DANIELA FENOGLIO (President)

GILBERTO FILACI

LESSONS

LESSONS START

As scheduled, lessons will be delivered in the second semester.

Class schedule

The timetable for this course is available here: Portale EasyAcademy

EXAMS

EXAM DESCRIPTION

The exam will consist of an oral examination in the presence of the course instructors. The interview will last approximately 25 minutes and will focus on the syllabus covered during lectures and laboratory sessions. Through discussion of various topics regarding the instrumentation, cytometric protocols covered, and the use of imaging, the commission will verify the achievement of the course's learning objectives.
At the end of the interview, the professors of the Commission will formulate a final vote which will derive from the overall judgment of the knowledge acquired by the candidate and from the clear exposition and appropriateness of the exposed arguments.

The basic elements of the subject must be known for the exam to be considered sufficient.
The grade of 30/30 with honors will be awarded when the knowledge / skills of the subject will be excellent.

ASSESSMENT METHODS

The oral exam will mainly focus on the topics covered during the lectures and the laboratories and will aim to verify the achievement of the appropriate level of knowledge and deepening of the topics covered. In particular, during the interview, the student will have to demonstrate the knowledge of: the anatomy of a cytometer and the fundamental principles of flow cytometry, the characteristics of the main fluorochromes, the guidelines in solving the compensation and related controls, the concepts of gating, the sample preparation protocols for the different types of cytometric and sorting measurements, guidelines in the acquisition and analysis of samples, the main applications of the cytometric platform in biology and medicine.

The final evaluation will also take into account the quality of the presentation, the correct use of specialist vocabulary and the ability to reason critically.

The basic elements of the subject must be known for the exam to be considered sufficient.

During the exam, the rules regarding the evaluation method of students certified as DSA or with other educational needs indicated by the university will be respected. (e.g.: additional time in the case of written exams, use of concept maps to be viewed for approval by the teachers at least one week before the exam date)

FURTHER INFORMATION

Regular class attendance is strongly recommended.

Contact the teacher for further information not included in the course information sheet.