Principles of Flow CytometryPrinciples of Flow Cytometry

Mohamed Labib Salem, PhD Prof. of Immunology

Founder and Executive DirectorCenter of Excellence in Cancer Research

Tanta University, Egypt

Cell phenotype/genotype

– Cell numbers – Cell size – Cell subsets– Cell apoptosis– Cell necrosis– Cell cycle– Chromosome– Cell sorting

Cell function

• Cellular function– Proliferation– Activation

• Molecular features– Protein secretion–Metabolites– Signaling pathways–MHC/HLA

What do you need from your cells?What do you need from your cells?

How do you analyze your cells?How do you analyze your cells?

• Histology/HistopathologyHistology/Histopathology• HistochemistryHistochemistry• ImmunohistochemistryImmunohistochemistry• BiochemistryBiochemistry• Magnetic- activated cell sorting (MACS)Magnetic- activated cell sorting (MACS)• ELISAELISA• ELISASpotELISASpot• Genetically: Genetically: FISH, PCR, DNA sequencing• OmicsOmics– Proteomic– Genomic

Flow Cytometry: Is the technologyFlow Cytometry: Is the technologyQualitativeQualitative

QuantitativeQuantitative

Flow Cytometer: Is the equipmentFlow Cytometer: Is the equipment

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

Flow CytometryFlow Cytometry

• It is routinely used in the diagnosis of It is routinely used in the diagnosis of health disorders, especially health disorders, especially blood cancersblood cancers, , but has many other applications in but has many other applications in basic basic research, clinical practice and clinical research, clinical practice and clinical trials. trials.

• A common variation is to physically sort A common variation is to physically sort particles based on their properties, so as particles based on their properties, so as to to purify populations of interestpurify populations of interest..

Flow Cytometry: Flow Cytometry: DefinitionDefinition

• In biotechnology, flow cytometry is a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus.

• It allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second.

Flow Cytometry: Flow Cytometry: HistoryHistory

• The first flow cytometry device was disclosed in U.S. Patent 2,656,508, issued in 1953, to Wallace H. Coulter.

• Mack Fulwyler was the inventor of the today's flow cytometers in 1965 with his publication in Science.

• The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Göhde from the University of Münster and first commercialized in 1968/69 by German developer and manufacturer PartecPartec.

• The first FACS (Fluorescence-activated cell sorting) instrument from Becton Dickinson (1974), the ICP 22 (1975) from Partec/Phywe and the Epics from Coulter (1977/78).

Parameter Microscopy Flow CytometryMeans of Analysis Cells immobilized on microscope

slide, counted manuallyCells suspended in liquid stream,pass single file in front of laser

Means of Detection Eye ElectronicNumber of Cells Analyzed Hundreds Thousands-MillionsRate of Analysis 100 cells per minute 2000-5000 cells/secondSensitivity Low-only strong protein

expression can be detectedHigh-Weak protein expressioncan be clearly identified.

Data Type Qualitative-Cells scored as +/- Quantitative-Fluorescence ofeach cell individually scored

LASER

FLUORESCENCE DETECTOR

FLUORESCENCE DETECTOR

MICROSCOPY FLOW CYTOMETRY

Flow Cytometry: 5 Requirements

Single cell suspension

Antigen- antibody conjugate

Fluorochrome & Fluorescence

Flow Cytometer

Cell analysis software

Chromosomes

Nuclear membrane

Nucleus

Plasma membrane

Centrioles

Cytoskeleton Endoplasmic reticulum

Ribosomes

LysosomeChloroplasts

Mitochondria

Golgi apparatus

Research & Clinical applications of the instrument BD FACSCanto Applications

Flow Cytometry: 5 Steps

Cell Preparation

Staining using labeled-Abs

Cell Acquisition

Cell Analysis

Data interpretation

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

Flow Cytometry: Cells

Flow Cytometry: AntigensAntigens• Serve as cell identifiers • T-cells have surface antigens CD4 and CD8 on

their surface • B-cells have surface antigens CD19, CD20, etc

B cell

T cell

Cell surface antigens

Flow Cytometry: Antibodies

ANTIBODIES: Proteins used

by the immune system to neutralize foreign invaders

Recognize, through specific binding antigens

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: Labeling

ANTIBODIES: Labeling with

dyes

Flow Cytometry: Labeling

Flow CytometryFlow Cytometry

How it worksHow it works

Flow Cytometer: Components

Flow Cytometry: Components Flow Cytometer: Components

How it worksHow it works

https://www.youtube.com/watch?v=TDRhCWaYRsg

Flow Cytometer: TypesBD FACSCalibur

Moxi Flow™

Partec Flow Cytometer

BD FACSVantageBD FACSAria

BD FACSCanto II cytometer

Flow Cytometer: Types

Analysis of solid tumor

Analysis of blood

malignancies

Stem cell phenotypenumbers

Bacterialanalysis

Viral analysis

Reticulocyte analysis

Detection of Immunodeficie

ncy diseases

Detection of autoantibodies

BD FACSCanto ApplicationsBD FACSCanto Applications

Sperm analysis

Fungal/parasitic analysis

Research & Clinical applications of the instrumentConclusion

ApoptosisProliferation

Cell cycle

Chromosome Analysis

Mohamed Labib Salem, PhDProf. of ImmunologyFounder and Executive DirectorCenter of Excellence in Cancer Research Tanta University, Egypt