Center of Excellence in Cancer Research Principles of flow cytometry Tanta University Principles of flow cytometry (SAAI)

Supervisors: Prof. Mohamed Labib Salem, PhD Prof. of Immunology; Executive Director, Center of Excellence in Cancer Research, Tanta University 2. Prof. Mohamed Attia Saad, MD Prof. of Clinical Pathology; Director of Flow Cytometry Unit, Center of Excellence in Cancer Research, Tanta University.

Instructor : Mona F. Zidan, BCs Master Researcher; Research Assistant, Flow cytometry Unit, Center of Excellence in Cancer Research, Tanta University Approved by the ICGEB to attened the international theoretical and practical training course "Flow cytometry and cell sorting in Biotechnology and Biomedicine Research" which was held at institut Pasteur de Montevideo, Montevideo, Uruguay, Latin America

3. Acquisition process 4. Analysis of data SAAI: Main objectives By the end of this session you will learn: Basics of flow cytometry 2. Staining process 3. Acquisition process 4. Analysis of data 5. Interpretation of data

Acquisition process Analysis of data SAAI: Main objectives By the end of this session you will learn: Basics of flow cytometry Staining process Acquisition process Analysis of data Interpretation of data

Flow Cytometry: Definition Flow ~ in motion Cyto ~ cell Metry ~ measure Measuring properties of single cells in a fluid stream Gives us the ability to analyze many properties of many cells in very little time

Three main basis: Flow Cytometry: Basis Single cell suspension Antigen- antibody conjugation Fluorochrome & Fluorescence

Three main basis: Flow Cytometry: Basis Single cell suspension Antigen- antibody conjugation Fluorochrome & Fluorescence

Flow Cytometry: Basis You need to have the cells flow one-by-one into the cytometer to do single cell analysis

Three main basis: Flow Cytometry: Basis Single cell suspension Antigen- antibody conjugation Fluorochrome &Fluorescent

Flow Cytometry: Basis Antigens Serve as cell identifiers T-cells have surface antigens CD4 and CD8 on their surface B-cells have surface antigens CD19, CD20, etc Antigens Cell surface antigens B cell T cell

Monoclonal Antibodies Polyclonal Antibodies Flow Cytometry: Basis Proteins used by the immune system to neutralize foreign invaders Recognize, through specific binding antigens Antibodies Monoclonal Antibodies Polyclonal Antibodies

Monoclonal antibodies Polyclonal antibodies Flow Cytometry: Basis Monoclonal antibodies Immunochemically identical antibodies React with a specific epitope on a given antigen Polyclonal antibodies Immunochemically dissimilar antibodies React with various epitopes on a given antigen

Antigen- antibody conjugation Cell surface antigen receptors Flow Cytometry: Basis Antigen- antibody conjugation Has many uses in the laboratory, including the specific identification of cells Surface Staining: Direct Conjugation Cell surface antigen receptors Cell A B + Incubation

Three main basis: Flow Cytometry: Basis Single cell suspension Antigen- antibody conjugation Fluorochrome & Fluorescence

Flow Cytometry: Basis Fluorochrome Fluorescent Molecule gives out a particular color when specific laser light hits it Fluorescent Molecule loses energy and emits light

Flow Cytometry: Basis

Flow Cytometer: Components Fluidics Optical system Electronics

Flow Cytometer: Components

Flow Cytometer: Components Flow Cytometry: Components

Flow cytometer: Light Scatter

Flow Cytometry: Process function Detection of: Phenotype Cell cycle Apoptosis function

Flow cytometry: Measurable Parameters Enzymatic activity pH, intracellular ionized calcium, magnesium Membrane fluidity Apoptosis Cell viability Monitoring electropermeabilization of cells Oxidative Burst Characterizing multidrug resistance (MDR) in cancer cells Glutathione Various combinations (DNA/surface antigens etc.)

Flow Cytometer: Types BD FACSCalibur Partec Flow Cytometer BD FACSAria BD FACSVantage Moxi Flow™

BD FACSCanto II cytometer Flow Cytometer: Types BD FACSCanto II cytometer

3. Acquisition process 4. Analysis of data Basics of flow cytometry Main objectives: SAAI By the end of this session you will learn: Basics of flow cytometry 2. Staining process 3. Acquisition process 4. Analysis of data 5. Interpretation of data

Staining process

Staining Process: Main components Take place in the processing Lab Equipments   Antibodies Reagents

Staining Types 1. Cell based staining Surface staining Intracellular 2. Cell free staining Sample should be converted into single cell suspension prior to staining and acquisition

Staining Process: Surface staining protocol Peripheral blood sample collected in EDTA tube Cells now are ready for acquisition

+ Surface Staining: Direct Conjugation Staining Process: Antigen- antibody conjugation Surface Staining: Direct Conjugation Cell surface antigen receptors Cell A B + Incubation

CD4 helper & CD8 Cytotoxic Staining Process: Antigen- antibody conjugation CD4 helper & CD8 Cytotoxic CD: Cluster of Differentiation The percentage of CD4 : CD8 = 2:1

3. Acquisition process 4. Analysis of data Basics of flow cytometry Main objectives: SAAI By the end of this session you will learn: Basics of flow cytometry Staining process 3. Acquisition process 4. Analysis of data 5. Interpretation of data

Acquisition process

Acquisition Process Takes place in acquisition Lab Three main steps: Pre-acquisition Acquisition Post acquisition

Acquisition Process Pre- acquisition: Turn on the system Open acquisition software Run warming & priming of the system (Fluidics start up) Make sure that priming process done successfully System now is ready for acquisition

Acquisition Process 2. Acquisition: Create a file with your exp. name Input your experiment layout Load your samples in the carousel (autoloader) Run the carousel and observe acquisition of the events on the worksheet

Acquisition Process Use different gating strategies, to capture your interested population (monocytes, etc.) from total cell population Select parameters to display (CD14 vs. CD33) Once acquisition is complete, export your experiment data as FSC extensions

3. Post acquisition: Run (Fluidics shut down) of the system Acquisition Process 3. Post acquisition: Run (Fluidics shut down) of the system Now system is safe to shut down

Acquisition Process Pre- acquisition:

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process 2. Acquisition:

Acquisition Process

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Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process

Acquisition Process 3. Post acquisition:

Acquisition Process

Acquisition Process

Questions

Acquisition process 4. Analysis of data Basics of flow cytometry Main objectives: SAAI By the end of this session you will learn: Basics of flow cytometry Staining process Acquisition process 4. Analysis of data 5. Interpretation of data

Analysis of data

Analysis Process: Main Steps The computer programs can be used to analyze data subsequent to its acquisition There are a variety of programs supplied for this purpose according to software acquisition Whatever the program used, the principles of data analysis are the same

Analysis Process: FlowJo software FlowJo is an open system for data analysis regardless acquisition software or data files extensions

Analysis Process: Light scatter FSC vs. SSC Forward scatter (FSC) Side scatter (SSC) measures scattered light at 90 degrees to the laser path and measures the granularity of the cell measures scattered light in the direction of the laser path and measures the size of the cell

Analysis Process : Gating strategy

Analysis process: Gating strategy SSC-H FSC-H

Analysis process: Gating strategy Anti CD8 Anti CD4

Analysis Process: Gating strategy

Basics of flow cytometry Acquisition process Analysis of data Main objectives: SAAI By the end of this session you will learn: Basics of flow cytometry Staining process Acquisition process Analysis of data Interpretation of data

Interpretation of data

Value of flow cytometric data interpretation Detection of T-cell subsets CD8 Cytotoxic T cell CD4 Helper T cell

Data Interpretation SSC-H FSC-H

Data Interpretation Anti CD8 Anti CD4

Questions

Practical Activities If you can, bring your lab coat Gp A 10.00 a.m Gp B 10.30 a.m Gp C 11.00 a.m Gp D 11.30 a.m Gp E 12.00 p.m Gp F 12.30 p.m If you can, bring your lab coat