basics of flow cytometry by prof. mohamed labib salem
TRANSCRIPT
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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
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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?
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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
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Flow Cytometry: Is the technologyFlow Cytometry: Is the technologyQualitativeQualitative
QuantitativeQuantitative
Flow Cytometer: Is the equipmentFlow Cytometer: Is the equipment
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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
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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..
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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.
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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).
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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
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Flow Cytometry: 5 Requirements
Single cell suspension
Antigen- antibody conjugate
Fluorochrome & Fluorescence
Flow Cytometer
Cell analysis software
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Chromosomes
Nuclear membrane
Nucleus
Plasma membrane
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Centrioles
Cytoskeleton Endoplasmic reticulum
Ribosomes
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LysosomeChloroplasts
Mitochondria
Golgi apparatus
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Research & Clinical applications of the instrument BD FACSCanto Applications
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Flow Cytometry: 5 Steps
Cell Preparation
Staining using labeled-Abs
Cell Acquisition
Cell Analysis
Data interpretation
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You need to have the cells flow one-by-one into the cytometer to do single cell analysis
Flow Cytometry: Cells
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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
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Flow Cytometry: Antibodies
ANTIBODIES: Proteins used
by the immune system to neutralize foreign invaders
Recognize, through specific binding antigens
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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
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Flow Cytometry: Labeling
ANTIBODIES: Labeling with
dyes
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Flow Cytometry: Labeling
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Flow CytometryFlow Cytometry
How it worksHow it works
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Flow Cytometer: Components
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Flow Cytometry: Components Flow Cytometer: Components
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How it worksHow it works
https://www.youtube.com/watch?v=TDRhCWaYRsg
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Flow Cytometer: TypesBD FACSCalibur
Moxi Flow™
Partec Flow Cytometer
BD FACSVantageBD FACSAria
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BD FACSCanto II cytometer
Flow Cytometer: Types
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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
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Mohamed Labib Salem, PhDProf. of ImmunologyFounder and Executive DirectorCenter of Excellence in Cancer Research Tanta University, Egypt