Cytometry - MB151P80E
Title: Cytometry
Czech title: Cytometrie
Guaranteed by: Department of Cell Biology (31-151)
Faculty: Faculty of Science
Actual: from 2024
Semester: summer
E-Credits: 4
Examination process: summer s.:
Hours per week, examination: summer s.:2/1, C+Ex [HT]
Capacity: 18
Min. number of students: unlimited
4EU+: no
Virtual mobility / capacity: no
State of the course: taught
Language: English
Explanation: The course cannot be combined with MB151P80.Please, enroll in the Moodle course (e-learning) - bottom right link.
Note: enabled for web enrollment
Guarantor: MVDr. Jozef Janda, Ph.D.
Incompatibility : MB151P80
Is incompatible with: MB151P80
Opinion survey results   Examination dates    E-learning course
Annotation -
You will be guided through the basic classification of cytometric methods in their historical context. Technological advantages and disadvantages of individual branches of cytometry will be documented using applications from various fields of science: botany, microbiology, protistology, hydrobiology, molecular biology, genetics, immunology and biomedicine in general.

Practical training is an integral part of this course and it is organized in 4 modules (3 hours each):
- Basic data analysis
- Experiment design
- Sample preparation and measurement of multi-parametric data
- Reporting and advanced data analysis

Applications of cytometry cover wide range of experimental approaches: analysis and sorting of particles of different sizes from sub-micron (chromosomes, bacteria, organelles, vesicles, exosomes) through cells (up to hundreds of micrometers) up to whole organisms of 1mm size. Analysis is not limited to static single parameter - univariate tests, but the main power of cytometry lies in multi-parametric analysis of heterogeneous samples and their functional properties. The introduction of new fluorochromes, including variants of fluorescent proteins, allows us to study functional processes in single living cells down to single protein-protein interactions. Therefore, cytometry is an essential building block of systems biology able to measure dozens of parameters in millions of cells at once.

Cytometry instruments as well as cytometry data are highly complex, fully standardized, and allow the use of robust statistical methods for the analysis. The course contains practical demonstration and detailed exercise in data analysis as well. The main emphasis is put on flow cytometry. Other variants, including mass or image cytometry, are covered theoretically but are not part of the practical exercise.

After completing the course you will have:
- Knowledge of principles of cytometry
- Knowledge of cytometry instrumentation and the use of fluorochromes and probes
- Knowledge of current applications in cytometry in the wide range of scientific fields
- Ability to assess the suitability of selected method in solving a specific problem or a question
- Ability to analyze and interpret results generated by flow cytometry
- Ability to critically assess the quality of cytometry data in the scientific publications
Last update: Drbal Karel, RNDr., Ph.D. (31.08.2023)
Literature -

Beginners:
GIVAN, Alice. L.: Flow Cytometry: First Principles, Wiley-Liss, New York, 2001, ISBN-13: 978-0471382249
ORMEROD, Michael. G.: Flow Cytometry - A Basic Introduction, http://flowbook.denovosoftware.com/

Intermediate:
DARZYNKIEWICZ, Zbygniew et al.: Methods In Cell Biology, Vol. 103: Recent Advances in Cytometry, Part A, Academic Press, Amsterdam, 2011.
DOLEŽEL, Jaroslav et al.: Flow Cytometry with Plant Cells. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007, ISBN-13: 978-3527314874

Experts:
SHAPIRO, Howard. M.: Practical Flow Cytometry, 4th Edition. John Wiley and Sons, Inc., New York, 2003, ISBN-13: 978-0471411253, available: https://media.beckman.com/-/media/pdf-assets/ebooks/flow-cytometers-ebook-practical-flow-cytometry.pdf
Coursera: MOUNT SINAI: Experimental Methods in Systems Biology

Protocols, webinars:
ROBINSON, J. Paul. et al.: Current Protocols in Cytometry, John Wiley & Sons, New York, 2003, ISBN-13: 978-0471142959

An additional information about selected education videos and MOOC courses will be provided during the course.

Last update: Drbal Karel, RNDr., Ph.D. (31.08.2023)
Requirements to the exam -

The course itself and the examination is running in-person. A study material and additional information is available in LMS Moodle.

The course credit requirement:
- three homework tests during the course is done in Moodle. The limit is 50% points in total - this is the first requirement.
- a final multiple choice test during the examination day is done in Moodle. At least 50% point limit is the second requirement for the exam entry.

The three exam parts consist of:

1/ A Report of a common experiment run in the 3rd practical lesson - evaluation of the correct design, conducting the measurement and data analysis. Submit a document in the format of a project report to Moodle by the end of May (a week before the exam) = 45 points.
2/ Your Experimental design for your real or a fictive project - use an example from the literature, introduction to the broad scientific picture incl. citations, a correct selection of fluorochromes/probes incl. your cytometer choice and spectral analysis, justification of the method of data collection and analysis and a description of the expected results - a short project style - max. 4 A4 pages, 45 points.
3/ An optional Wikipedia entry (5000 bytes) in English on the topic in line with Cytometry, including 3-5 matching citations = max. 10 points / login to Wikipedia under your account which I can recognize and enroll in Wikipedia: https://outreachdashboard.wmflabs.org/.

The exam consists of three documents submitted until a week before the final exam = in this case you reach 100% points. There is a 2% progressive penalty for each day of delay from the previous day until the term of presentation during the last week in May/first week in June - to be discussed with all students.
According to the evaluation by the tutor, you are going to correct your work in a presentation within the last few days. All students will present their work in 10 minutes presentation of part 1 and 2 in front of all other students in-person. After each presentation, all students will discuss and evaluate each other. Yet, the final decision is up to the tutor, who is going to justify your grades based on your ranking and all other parts of the exam as well. Tutor's decision will be submitted in a written form in Moodle.

A good example of Report and Experimental design will be provided.

Classification: each part of the exam is evaluated separately, and the grading complies with Charles University rules:

    100-94 points = A (excellent - summa cum laude) =  1
    93-80 = B (very good - insigni cum laude); in Czech classification system = 1
    79-60 points = C (good - magna cum laude); in Czech classification system = 2
    59-47 points = D (quite good - cum laude); in Czech classification system = 3
    46-40 = E (pass - rite); in Czech classification system = 3
    below 39 = FX, F (fail); in Czech classification system = 4

In case of a borderline result (77 - 79, 57 - 59, 37 - 39) there is an oral examination done immediately during the presentation day.

Last update: Drbal Karel, RNDr., Ph.D. (31.08.2023)
Syllabus -

The Cytometry course is running in 4 subsequent cycles, each containing 3 lectures in two lecture hours (1,5 hours) and one 3-hour practical training which immediately follows the last lecture in each cycle the same day.


1. cycle: Cytometry basics
LECTURES:
1. Basics of cytometry - technology, terminology and history.
Classification of cytometry techniques - diverse methods of particle detection up to the cytomics (fluorescent, mass, image, -omics combinations). History, parameters measured by flow cytometry, main parts of flow cytometer - fluidics, laminar flow, flow cell, hydrodynamic focusing, principles of sorting.
Electronics and optics of cytometers - light sources, mercury lamps and lasers, optical filters. Detection of light pulses, photodiodes and photomultipliers, parameters of light pulse. Signal amplifiers, digital processing, fluorescence compensation, types of plots for data display.
2. Basic application of flow cytometry
Introduction to data analysis - nuclear DNA content, analysis of ploidy, size of nuclear genome, identification of interspecific hybrids, detection of aneuploidy, determination of mode of reproduction, analysis of cell cycle kinetics, apoptosis, analysis of gene expression, study of phytoplankton.

3. PRACTICAL TRAINING: Basics of practical data analysis
What you see is what you get: data quality, data standards, data quality, data filtering, resolution and data distribution, transformation, displaying the data - basic plots. Introduction to the open source software for basic analysis (FCSalyzer, opt. Flowing Software), complex software packages (SW: FlowJo, BC Kaluza, BD DiVa).

Course credit test #1

2. cycle: Basic cytometric applications
LECTURES:
4. Fluorochrome principles and the use of cytometry in biotechnology and biomedicine:
Previous cycle repetition with the focus on standardization and reproducibility of measurement. Small fluorochromes, tandem dyes, FRET, fluorescent proteins, sensors. Selection methods in biotechnology, environmental analysis, connection to the -omics methods.

5. Correlation of the measurement to the data visualization
Optics: 3 planes in the flow cell, interrogation point, laser delay, fluorochrome excitation/emission (filters, dichroic mirrors), detectors and signal amplifiers, spectra viewers.
Data analysis: light pulse parameters and detection, digital signal processing, compensation of spectral overlap, 1D, 2D, 3D, 4D-kinetics, multiD analysis (incl. dimensionality reduction principles).

Functional tests in cytometry:
Apoptosis, proliferation, cell signaling, mitochondria, membrane potential, kinetic tests, rare cells, metabolism. Immunopenotyping of cells and tissues: immune response, tumors, infections, microvesicles, microparticles.
Opt. - cytometry methods in virology, microbiology, hydrobiology, protistology and ecology.

6. PRACTICAL TRAINING: Experimental design
A design of an experiment (to be done in the 3rd practical lesson) and the limitations of cytometry methods (statistical background - rare cell, autofluorescence, compensation, basics of combinations of fluorochromes, design of antibody panels). Sample preparation theory (solid tissues, bacteria, yeast, viruses, biosafety regulations)

3. cycle: Details of analysis and data measurement
LECTURES:
7. Practical data analysis tutorial (titration/voltration)
An example of titrated data analysis followed by the measurement of titrated samples.

8. Working with real data
SI calculation based on the data measured last lesson, preparation of a report.

Course credit test #2

9. PRACTICAL TRAINING: Sample preparation and measurement of multiparametric data
Preparation of 8-color panel of monoclonal antibodies according to the recommendation of EUROFLOW, labeling of blood samples and measurement on a multi-parametric flow cytometer (8-color FACSVerse plus an additional one having more detectors = LSR-II, CytoFLEX S).

4. cycle: Multiparametric data analysis
LECTURES:

10. Basic principles of multiparametric data analysis (FlowJo introduction)
Data compensation, visualization, scaling, transformation, normalization. Population identification, FMO controls and controls in general, file concatenation, graphical and statistical output in FlowJo.

11. Data compensation tutorial (FlowJo)

12. PRACTICAL TRAINING: Work with our measured data
The use of FlowJo in supervised analysis and R tools for unsupervised analysis, data quality check, a complete process of data evaluation, reporting and general troubleshooting - exam training.

Course credit test #3

Last update: Drbal Karel, RNDr., Ph.D. (31.08.2023)