introduction to flow cytometry -- bd facscanto iitechcomm.lifescience.ntu.edu.tw/tcx/file/facscanto...
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Daisy KuoAssistant Product ManagerE-mail: [email protected]
Introduction to Flow Cytometry
-- BD FACSCanto IITM
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Outline
• Basic Concept of Flow Cytometry
• FACSCanto II System Introduction
• Application Examples
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What is Flow Cytometry?
• Flow = Fluid• Cyto = Cell• Metry = Measurement
• A variety of measurements are made on cells, cell organelles, and other objects suspended in a liquid and flowing at rates of several thousands per second through a flow chamber.
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What Can a Flow Cytometer Tell Us About a Cell?
Its relative size (Forward Scatter—FSC)
Its relative granularity or internal complexity (Side Scatter—SSC)
Its relative fluorescence intensity
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Subsystems
FluidicsTo introduce and focus the cells for interrogation.
OpticsTo generate and collect the light signals.
ElectronicsTo convert the optical signals to proportional digital signals, process the signals, and communicate with the computer.
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Sample Differential
Sample
High Sample Pressure
120 µL/min
High Differential Pressure
LaminarFlow
Sheath Sheath
LaminarFlow
Low Differential Pressure
Sheath SheathSample
Low Sample Pressure
12 µL/min
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Optics
• Excitation optics– Lasers Lenses to shape and focus the laser beam
• Collection optics– A collection lens to collect light emitted from the
article-laser beam interaction– A system of optical mirrors and filters to route
specified wavelengths of emitted light to designated optical detectors
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Excitation Optics
• Spatially separated laser beams lower the possibility of fluorescence spillover
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Optics-- ConfigurationOther Fluorochrome
GFP
PI
PI, PE-Cy5.5, 7-AAD
Alexa Fluor® 633
DAPI, Hoechst Dye
Cascade Blue®
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PMTs and preamps convert photons to voltage pulses.
Analog-to-digital converters translate analog signals to proportional digital signals.
Compute area and height for each pulse.
Perform compensation and calculate ratios and width.
An embedded computer interfaces with the computer workstation for data transfer.
Electronics
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Analog-to-Digital Converter
Baseline
16,364
Time
937
1,985
7,650
12,420
Hei
ght
351
383
375
406
377
318
367
319
375
423
432
937
1985
7650
1242
015
300
1325
657
9124
7184
243
333
131
130
837
634
941
482
313
7390
351
433
841
830
731
735
331
370
340
337
830
840
640
530
335
340
532
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Digitized values
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39,27139,271
Data Storage
Event 1Event 2Event 3
FSC SSC FITC PE0 60 120 89
10 160 65
30 650 160
List-Mode Data
39,271
89
675 30,621PE
FITC
675Time
PEFI
TC
39,271
22,688
30,621
6,189
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Spectral Overlap– Compensation Theory
APC PerCPPI
Wavelength (nm)400 500 600 700
100%
0%
Pacific Blue
Nor
mal
ized
Inte
nsity
FITC PEAmCyan PerCP-Cy5.5
800
PE-Cy7
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Spillover
FITC PE PerCP-Cy5.5
650 700
PerCP-Cy5.5695/40
500 600
FITC530/30
Rel
ativ
e In
tens
ity
Wavelength (nm)
550
PE585/42
PE-Cy7
PE-Cy7780/60
750 800
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FITC Spillover
650 700
PerCP-Cy5.5695/40
500 600
FITC530/30
Rel
ativ
e In
tens
ity
Wavelength (nm)
550
PE585/42
750 800
FITC
FITC
PEP
erC
P-C
y5.5
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650 700
PerCP-Cy5.5695/40
500 600
FITC530/30
Rel
ativ
e In
tens
ity
Wavelength (nm)550
PE585/42
FITC CompensationTo lower cluster, increase value.
FITC FITC
FITC
Per
CP
-Cy5
.5
FITC
Per
CP
-Cy5
.5
PE-%FITC
PerCP-Cy5.5-%FITC
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Compensation Examples
Correct Compensation Undercompensation Overcompensation
Incorrect Compensation
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Applications
• Phenotype Analysis (Cell Surface Antigens/Markers)• Intracellular Analysis
-- Eg. Cytokines, Signal Transduction molecules…etc.
• DNA Analysis-- Eg. Viability, Cell cycle, Apoptosis…etc.
• Cell Fuction Analysis-- Eg. Free radicals, Ca2+, Reporter genes…etc.
• CBA (Cytometric Bead Array)• Others
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Lymphocyte Immunophenotyping
Granulocytes
Monocytes
Eosinophils
Basophils
NeutrophilsLymphocytes
T B NK
T Helper
TCytotoxic
Peripheral White Blood CellsCD45+
CD3+
CD4+ CD3+
CD8+
CD3+
CD3-
CD19+
CD3-
CD16+
CD56+
Monocytes
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Permeabilizing solution
• Cytokine• Enzyme• signal transduction
molecule• …etc.
Intracellular Analysis
Fixation solution
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CD3CD28
PHA
Con A
IonomycinLPS
T cell
Stimulation
Fixation
Secretion stop(Brefeldin A or Monensin)
Only in vitro
PermeabilisationIntracellular Staining
To enhance the accumulation of intracellular cytokines.
Monensin: Cytokines accumulate in the ERBrefeldin A: in Golgi complex.
To maintain structural integrity.Formaldehyde or glutaraldehydeKeep the protein structure and doesn't change the(accessibility of the) epitopes too much
Saponin (permeablisation buffer).
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• Membrane Potential (DiOC6, JC-1)
• Oxidative Metabolism (Free Radicals)
• Intracellular PH Value (Snarf-1)
• Ca++ Influx (Fluo-4/Fura Red, Indo-1)
• Phagocytosis
• Cell Proliferation (PI, BrdU, Intracellular Cyclins)
• Apoptosis (Annexin V, active Caspase-3)
Cell Function Analysis
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C a++ C a++C a++
C a++
Externalization ofphosphatidylserine
Plasmamembrane
Apoptosis
Cytoplasm Cytoplasm
Annexin V-FITCconjugate
Annexin V Assay
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Beads Provide a Flexible Platform
Multiple sizes
Different fluorescence
intensities
Different colors with different intensities
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Advantages of Bead-Based Immunoassays• Analyze multiple analytes simultaneously• Reduced sample volume requirements• Reduced hands-on time by parallel analysis of samples• Wide dynamic range of fluorescence detection (requires
fewer sample dilutions)
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Proteins MeasuredA. Interleukin (IL)-2B. IL-4C. IL-5D. IL-10E. Tumor Necrosis Factor-F. Interferon-
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CBA Flex Sets• Open configuration (Up to 30 plex)• Clustering based on Red and NIR fluorescence intensity• Need to be used at dual-laser(488nm blue v.s 633nm red) instrument