principles of flow cytometry (saai) c enter of e xcellence in c ancer r esearch tanta university
TRANSCRIPT
Principles of flow cytometry(SAAI)
Center of Excellence in Cancer Research
Tanta University
Supervisors:
1. 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
By the end of this session you will learn:1.Basics of flow cytometry2. Staining process3. Acquisition process4. Analysis of data5. Interpretation of data
SAAI: Main objectives
By the end of this session you will learn:1.Basics of flow cytometry2.Staining process3.Acquisition process4.Analysis of data5.Interpretation of data
SAAI: Main objectives
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
Flow Cytometry: Definition
• 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
You need to have the cells flow one-by-one into the cytometer to do single cell analysis
Flow Cytometry: Basis
• Three main basis:
Flow Cytometry: Basis
Single cell suspension
Antigen- antibody conjugation
Fluorochrome &Fluorescent
Flow Cytometry: Basis
• Serve as cell identifiers • T-cells have surface antigens CD4 and CD8 on
their surface • B-cells have surface antigens CD19, CD20, etc
Antigens
B cell
T cell
Cell surface antigens
Flow Cytometry: Basis
Proteins used by the immune system to neutralize foreign invaders
Recognize, through specific binding antigens
Antibodies
Polyclonal AntibodiesMonoclonal Antibodies
Polyclonal antibodies• Immunochemically dissimilar
antibodies• React with various epitopes on a
given antigen
Monoclonal antibodies• Immunochemically identical antibodies• React with a specific epitope on a given
antigen
Flow Cytometry: Basis
Flow Cytometry: Basis
Cell
A
B + Incubation
Surface Staining: Direct Conjugation
Cell surface antigen receptors
Antigen- antibody conjugation
• Has many uses in the laboratory, including the specific identification of cells
• Three main basis:
Flow Cytometry: Basis
Single cell suspension
Antigen- antibody conjugation
Fluorochrome & Fluorescence
Flow Cytometry: Basis
Fluorochrome
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
Electronics
Optical system
Flow Cytometer: Components
Flow Cytometry: Components Flow Cytometer: Components
Flow cytometer: Light Scatter
Flow Cytometry: Process function
Cell cycle
Detection of:
Apoptosis
function
Phenotype
1. Enzymatic activity
2. pH, intracellular ionized calcium, magnesium
3. Membrane fluidity
4. Apoptosis
5. Cell viability
6. Monitoring electropermeabilization of cells
7. Oxidative Burst
8. Characterizing multidrug resistance (MDR) in cancer cells
9. Glutathione
10. Various combinations (DNA/surface antigens etc.)
Flow cytometry: Measurable Parameters
Flow Cytometer: TypesBD FACSCalibur
Moxi Flow™
Partec Flow Cytometer
BD FACSVantageBD FACSAria
BD FACSCanto II cytometer
Flow Cytometer: Types
By the end of this session you will learn:1.Basics of flow cytometry2. Staining process3. Acquisition process4. Analysis of data5. Interpretation of data
Main objectives: SAAI
Staining process
Staining Process: Main components
• Take place in the processing Lab
Equipments
Antibodies Reagents
Staining Types
1. Cell based staining
Surface staining
Intracellularstaining
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
Staining Process: Antigen- antibody conjugation
Cell
A
B + Incubation
Surface Staining: Direct Conjugation
Cell surface antigen receptors
CD4 helper & CD8 Cytotoxic
• CD: Cluster of Differentiation• The percentage of CD4 : CD8 = 2:1
Staining Process: Antigen- antibody conjugation
By the end of this session you will learn:1.Basics of flow cytometry2.Staining process3. Acquisition process4. Analysis of data5. Interpretation of data
Main objectives: SAAI
Acquisition process
Acquisition Process
• Takes place in acquisition Lab
• Three main steps:
1. Pre-acquisition
2. Acquisition
3. Post acquisition
Acquisition Process1. Pre- acquisition:
Acquisition Process
Acquisition Process
Acquisition Process
Acquisition Process
2. Acquisition:
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
Acquisition Process
Acquisition Process3. Post acquisition:
Acquisition Process
Acquisition Process
Quest
i
ons
By the end of this session you will learn:1.Basics of flow cytometry2.Staining process3.Acquisition process4. Analysis of data5. Interpretation of data
Main objectives: SAAI
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
Side scatter
(SSC)
Forward scatter (FSC)
measures scattered light in the direction of the laser
path and measures the size of the cell
measures scattered light at 90 degrees to the laser path and measures the granularity of the cell
FSC vs. SSC
Analysis Process : Gating strategy
Analysis process: Gating strategy
FSC-H
SSC-
H
Anti CD4
Anti
CD8
Analysis process: Gating strategy
By the end of this session you will learn:1.Basics of flow cytometry2. Staining process3.Acquisition process4.Analysis of data5. Interpretation of data
Main objectives: SAAI
Interpretationof data
Data Interpretation
Value of flow cytometric data interpretation
Detection of T-cell subsets
CD8 Cytotoxic T cellCD4 Helper T cell
Data Interpretation
FSC-H
SSC-
H
Data Interpretation
Anti CD4
Anti
CD8
Quest
i
ons
Practical Activities
Gp A10.00 a.m
Gp B10.30 a.m
Gp C11.00 a.m
Gp D11.30 a.m
Gp E12.00 p.m
Gp F12.30 p.mIf you can, bring your lab coat