Flow Cytometry Flow Cytometry (Principles and Main Applications) Hossein Asgarian-Omran Hossein Asgarian-Omran Ph.D., Immunology Dep. of Immunology, School of Medicine Mazandaran University of Medical Sciences Mazandaran University of Medical Sciences [email protected]
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Hossein AsgarianHossein Asgarian-OmranFlow CytometryFlow Cytometry (Principles and Main Applications) Hossein AsgarianHossein Asgarian-Omran Ph.D., Immunology Dep. of Immunology, School
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Flow CytometryFlow Cytometry(Principles and Main Applications)
How to interpret flow cytometry-related results andHow to interpret flow cytometry related results andgraphs in scientific documents
How to use this system in your own projects
HistoryThe first fluorescence‐based flow cytometry device (ICP 11):by Wolfgang Göhde (University of Münster, Germany. 1968)
The first commercialized by German developer andmanufacturer Partec in Göttingen (1968‐69)g ( )
The first FACS instrument from Becton Dickinson (1974)
Original name of flow cytometry technology was: "pulsecytophotometry"cytophotometry
8 years later in 1976, at the Conference in Florida, the namewas changed to "flow cytometry", a term that quicklybecame popular.
Definition
Flow Cytometry
Flow Cytometry is the process whereby such measurements aremade upon cells/particles as they pass through a measuring apparatussuspended in a fluid stream.
• Individual cell fluorescence quanta is picked up by the variousdetectors (Photo Multipliers Tubes, PMT’s).detectors (Photo Multipliers Tubes, PMT s).
• PMT’s convert light into electrical pulses and amplify them.
• These electrical signals are digitized using Analog to DigitalConverters (ADC’s).
• Different parameters are determined for each single event.
Different Parameters Measured by Flow Cytometryy y y
Intrinsic Extrinsic
• No reagents or probes • Reagents are required.
Intrinsic Extrinsic
g prequired (Structural)
ll i ( d i h
‐ Mostly fluorescent probes
– Cell size (Forward Light Scatter)
– Cytoplasmic grabularity (90 d Sid Li h S )degree Side Light Scatter)
Forward Angle Light Scatter or FSCForward Angle Light Scatter or FSC
Laser
FALS Sensor
Laser
90 Degree Light Scatter or SSC
FALS Sensor
Laser
90LS Sensor
Fluorophores in Flow Cytometry
Common FluorophoresCommon Fluorophores
• Organic: FITC, APC, PE, PerCP
• Tandem: PECY7, PECY5.5, APC‐tandem
• Nanocrystal: Q‐dot(Invitrogen), eFluor y g(eBiosciences) and Brilliant Violet (Biolegend and BD Biosciences)
Common Fluorophores in Flow Cytometry u p w y y
400 nm 600 nm 700 nm
ty
Wavelength
500 nm
E it ti
e In
tens
it ExcitationEmission
Rel
ativ
e
Fluorescein (FITC)Fluorescein (FITC)
Fluorochromes (cont…)
W l th
Fluorochromes (cont…)
400 nm 600 nm 700 nm
ity
Wavelength
Excitation500 nm
ve In
tens
i
Emission
Rel
ativ
Phycoerythrin (PE)Phycoerythrin (PE)
• Each cell generates a quanta of fluorescencePhotomultiplier Tubes
Flow Cytometry in Different EraFlow Cytometry in Different Era
• Immunology and hematologygy gy
• Cell Kinetics
• Genetics
• Molecular Biology
• Pharmacology
• Microbiology
• Parasitology
• Animal Husbandry (and Human as well)• Animal Husbandry (and Human as well)
• Biological Oceanography
• BioterrorismBioterrorism
Applications of Flow CytometryDetermination of cellular PhenotypesAbsolute cell countingImmunophenotyping of leukemia and lymphomaImmunophenotyping of leukemia and lymphomaMeasurement of intracellular and nuclear antigens (cytokines)Functional assays by flow cytometry (phagocytosis) Analysis of cell division using CFSEAnalysis of cell division using CFSEAnalysis of reticulocyte Staining of microbial cellsDNA analysisChromosome analysis and sortingApoptosisApoptosisFlow cytometric screening of cell‐based librariesMultiplexed particle based flow cytometric assaysCell sortingCell sortingand …..
Functional Assays by Flow CytometryFunctional Assays by Flow Cytometry
• Phagocytosis• Oxidative metabolism• Hydrogen peroxide production• Glutathione levels• Degranulation assays• Ion flux measurement of cytosolic free Ca• Detection of dead cells and measurement of cell killikilling
ImmunophenotypingImmunophenotyping
• Detecting the origin/stage of differentiation ofDetecting the origin/stage of differentiation of leukemia/lymphoma
• Detecting early recurrence of hematological g y gmalignancies
• Diagnosis/monitoring inherited/congenital g g gimmunodeficient patients
• Chemotherapeutic monitoring
• Diagnosis/monitoring autoimmune disease
• Pre/post transplantation monitoring/evaluationp p g
DNA AnalysisDNA Analysis
GG22MM GG00
DNA AnalysisDNA AnalysisGG11
ssGG00GG11ss
Cou
ss GG22MMnt
0 200 400 600 800 1000
DNA content22NN 44NN
DNA content
Staining of microbial cells•Real time analysis •Measurement of total cell count•Monitoring of microorganisms in food, water and …•Sorting and identifications of bacterial spores
B.anthracis
B.subtilisSS
irradiated B.anthracis
SS
FS
Reticulocyte Analysis
•Erythropoietin therapeutic effect•BMT recovery
50
BMT recovery•Evaluation of erythropoiesis
15
112
Cou
nt
75
RR11RR22RR33RR44
1000
0
37
Thiazole Orange.1 1000100101
Apoptosis
Dual Staining with Annexin V and PIua Sta g t e a d
Multiplexed particle based flow t t icytometric assays
-The technology utilizes microspheres as the solid support for a conventional immunoassay affinity assay or DNA hybridization assayconventional immunoassay, affinity assay or DNA hybridization assay.
Ab against microorganismsCytokine
Various protein DNA and RNAVarious protein, DNA and RNAViral antigens