principles of flow cytometry (saai) c enter of e xcellence in c ancer r esearch tanta university

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