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

Attendance and participation in training activities (lectures and laboratory attendance) will allow the student to acquire the necessary knowledge to deal with the different types and approaches that apply cytometric measurements and sorting in the biological field.
Specifically, the course aims to achieve the following objectives:
- knowledge of the fundamental principles of flow cytometry and instrumentation;

- reference points and guidelines in the execution of cytometric measurements;

- deepening of applications for studies in the biological field;

- reading and interpretation of the results obtained.

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 together with other bibliographic material.

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 test in the presence of the course teachers. The interview will last about 25 minutes per teacher and focus on the program carried out during the frontal lessons and the laboratori lessons. Through the discussion of various topics and the use of images of cytometric measurements, the Commission will verify the achievement of the educational objectives set by the teaching.
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 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.

Exam schedule

FURTHER INFORMATION

Regular class attendance is strongly recommended.