Download Application of Flow Cytometry

Flow Cytometry
Basic Introduction and Principle
Cloe Chang 張舒婷
Product Specialist
BD Bioscience
[email protected]
Sep 12, 2016
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1
Topic
 Introduction of Flow Cytometry
 Instrument setup
 Application of Flow Cytometry
2
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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.
Confidential—For Internal Use Only
Particle Size
• Detection range: 0.5~50m
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What Can a Flow Cytometer
Tell Us About a Cell?
• Its relative size (Forward Scatter—FSC；前向散射光)
• Its relative granularity or internal complexity
(Side Scatter—SSC；側向散射光)
• Its relative fluorescence intensity
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Scatter Light
Forward
Laser
FSC Sensor
o
90
SSC Sensor
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Side Scatter (SSC)
Ex. Lysed Whole Blood
Neutrophils
Monocytes
Forward Scatter (FSC)
Lymphocytes
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Fluorescence Light
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Fluorescence intensity
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BD Flow Cytometers – Cell Analyzer
FACSCalibur
FACSCanto
2 Lasers,
4 Colors Analyzer
2 Lasers,
6 Colors Analyzer
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BD Flow Cytometers – Cell Sorter
FACSAria
3 Lasers,
11 Colors Sorter
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Main Component
Fluidics 液流系統
To introduce and focus the cells for interrogation.
Optics 光學系統
To generate and collect the light signals.
Electronics 電子系統
To convert the optical signals to proportional digital signals,
process the signals, and communicate with the computer.
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Fluidics Pressurized System
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Sample Flow
Excitation Lasers
Hydrodynamic
Focusing
Flow Cell
sheath
sheath
sample
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Sample Differential
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Optical
 Excitation optics:
 Lasers: 488nm(Blue) + 633nm(Red)
 Filters and mirrors that route the laser light to the
fluid stream
 Collection optic:
 Fiber optic cables that direct the emitted light to the
appropriate emission block
 Filters that direct the signals in the emission block to
the appropriate photomultiplier tube (PMT)
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Excitation and Emission
 Use the maximum excitation wavelengths to determine
lasers that can be used to excite the fluorochrome.
 Use the maximum emission wavelengths to determine
filters and PMTs that can be used to measure the signal.
488
530/30
Emission
Excitation
FITC
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FACSCalibur Optics
Collection optics
Excitation optics
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Optical Filters
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FACSCalibur Optics
FL1
530/30
SSC
FL2
FL4
Collection Optics
488/10
585/42
90/10 Beam Splitter
661/16
DM 560SP
DM 640LP
670LP
Half Mirror
Excitation Optics
488 nm
Blue Laser
Fluorescence
Collection Lens
FL3
Flow
Cell
~635 nm
Red Diode Laser
488/10
Focusing
Lens
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FSC Diode
FACSCalibur Configuration
488nm
635nm
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FACSCanto Excitation Optics
633nm
488nm
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FACSCanto Collection Optics
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Collection Optics—Octagon
Blue Laser Signal
• 接收光源順序：能量弱→強
750–810 nm
• 反射傳遞：能量折損較低
564–606 nm
515–545 nm
483–493 nm
> 670 nm
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Collection Optics—Trigon
Red Laser Signal
650–670 nm
750–810 nm
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FACSCanto —Octagon and Trigons
(BD專利設計)
因為專利光學設計，使FACSCanto
流式細胞儀靈敏度及解析度皆提升!
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BD FACSCanto Configuration
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Electronics
 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.
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Pulse Height
Laser
Volts
Creation of a Voltage Pulse
Time (µs)
Pulse Width
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Quantification of a Voltage Pulse
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Data Storage
Histogram
List-Mode Data
Time FSC SSC
FITC
PE
Event 1
0
60
120
675
39,271
39,271
89
Event 2
10
160
65
39,271 30,621
Event 3
30
650
160
22,688
6,189
Dot Plot
FITC
FITC
39,271
89
675
PE
30,621
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PE
Data Display
 Linear Scaling
 Log Scaling
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Linear vs. Log
Volts
FL1 Voltage Pulses
Linear mode
0.1 V
Volts
Time
0.4 V
Pulse Height
0V
10 V
Volts
Time
1.2 V
Log mode
Volts
Time
4V
Volts
Time
8V
0.001 V
Time
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0.01 V
0.1 V
Pulse Height
1V
10 V
Linear v. Log Amplification
 Linear amplification is usually used for light scatter parameters
and DNA analysis.
 Log amplification is used for fluorescence signals with a large
dynamic range.
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Flow Cytometry Detection Principle
Time
SSC
1. fluidics
Time
FSC
Time
PE
Data
Processing
FITC
APC
Time
Time
2. optics
PerCP-Cy5.5
Time
Computer operation
3. electronics
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Topic
 Introduction of Flow Cytometry
 Instrument setup
 Application of Flow Cytometry
36
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Auto-fluorescence
Non-stain sample
UL
UR
LL
LR
Auto-fluorescence
After voltage adjustment
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Compensation theory
Emission Optics
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FITC Spillover
PE
585/42
Relative Intensity
FITC
530/30
500nm
550nm
600nm
650nm
Wavelength (nm)
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700nm
FITC Compensation
To Lower Cluster
Increase %
Subtracted
PE
585/42
Relative Intensity
FITC
530/30
20.10% of the Signal from
FITC Sensed in PE
500nm
550nm
600nm
650nm
Wavelength (nm)
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700nm
FITC Compensation
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Overcompensation vs Undercompensation
Correct
Compensation
Overcompensation
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Undercompensation
Compensation Examples
Incorrect Compensation
Correct Compensation
Undercompensation
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Overcompensation
Topic
 Introduction of Flow Cytometry
 Instrument setup
 Application of Flow Cytometry
44
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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)
-- cytokine detection
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Phenotype Analysis
 Ligand
 Receptor
 Adhesion molecule
 …etc
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Lymphocyte Immunophenotyping
Peripheral White Blood Cells
CD45+
Monocytes
Granulocytes
Lymphocytes
Monocytes
Neutrophils
B
T
CD3+
T
Helper
CD3+
CD4+
Confidential—For Internal Use Only
T
Cytotoxic
CD3+
CD8+
CD3CD19+
HLA-DR+
NK
CD3CD16+
CD56+
Basophils
Eosinophils
Lymphocyte Subset
48
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Intracellular Analysis
 Cytokine
 Enzymes
 Structure Proteins
 Intracellular Signaling
Confidential—For Internal Use Only
Intracellular Analysis
Permeabilizing
solution
Fixation
solution
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Cell Surface and Cytoplasmic
Stain protocol
Confidential—For Internal Use Only
Detection of Cell Surface and
IC Cytokine
CD4 FITC
CD4 FITC
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CD4 FITC
Cell Function Analysis
 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)
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Cell Apoptosis 細胞凋亡
－ Annexin V Apoptosis Assay
PS
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Annexin V/PI Double Staining
Early
Apoptosis
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Cell Apoptosis 細胞凋亡
－Mitochondria potential change-JC-1 (BD Mitoscreen)
Apoptosis通常與mitochondria的膜電位(Δψ)去極化有關性
 JC-1 = J-aggregate-forming Cationic Δψ sensitive dye
J-aggregates:膜電位Δψ 極化(polarized)時為此型式
JC-1 monomers: 膜電位Δψ (depolarized)去極化時存在此型式
J-aggregates 存於健康細胞，且螢光為FL1及FL2 channels (綠色及橘紅色螢光)
JC-1 monomers通常但非絕對存於Δψ 去極化的apoptosis細胞，且只剩下FL1 (綠色螢光)
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Cell Apoptosis 細胞凋亡
－ JC-1(BD Mitoscreen) Jurkat T cell data
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Cell Apoptosis 細胞凋亡
－ MitoStatus TMRE/Red
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DNA Analysis
Ethanol
Detergent
Nucleic Acid Dye
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DNA Dye
Propidium
NH2
7-AAD
NH2
N+
C2H5
C2H2)3N+
CH3
C2H5
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Cell Cycle Analysis
G2
M
G0
G0 G1
G1
S
Count
S
0
200
400
G2 M
600
4N
2N
DNA content
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800
1000
Apoptosis (Sub G1)
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Application of Flow Cytometry
 Soluble form protein detection
Confidential—For Internal Use Only
Cytometric Bead Array (CBA)
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Beads Provide a Flexible Platform
Different fluorescence
intensities
Different colors with different intensities
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Cytometric Beads Array (CBA)
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100
10
Simultaneous Analysis of Multiple Cytokines
1
10000
IL-2
IL-4
IL-5
IL-10
TNF-a
IFN-g
1000
1000
100
100
10
10
1
1
10000
1000
1000
1000
100
100
100
10
10
0 pg
1
20 pg
1
10
40 pg
1000
100
100
10
1
312 pg
625 pg
10
10
100
1000
1
1250 pg
2500 pg
10
100
1000
5000 pg
1
11
1000
156 pg
1
1
1000
80 pg
10
10
100
100
1000
1000
11
10
10
10
100
100
100
1000
1000 10000
1000
10
100
1000 10000
FL2
100
10
1
10 trademarks
100 1000
1000 are property of Becton, Dickinson
11 all other
10
100
1
10 Company.
100 1000 10000
1
© 2016 BD. BD, the BD Logo and
and
10
100
1000
FL3
Standard Curves
IL-2 PE MFI:567
IL-2: 625pg/ml
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BD Pharmingen Product list
Cat#
Name
BD Pharmingen™
550285 PI/RNase Staining Buffer
556463
BD Pharmingen™ Propidium
Iodide Staining Solution
BD Pharmingen™
556419 Annexin V
556547
Size
The reagent is suspended in
a phosphate-buffered
solution (pH 7.2) with 0.02%
(w/v) sodium azide
100ml
Propidium Iodide Staining
Solution
(For AnnexinV/PI assay use)
2ml
Annexin V-FITC
200 test
BD Pharmingen™ Annexin V Annexin V-FITC, Propidium
: FITC Apoptosis Detection
Iodide Staining Solution,
Kit I
Annexin V Binding Buffer
100 test
BD Pharmingen™
100 test
551302 BD™ MitoScreen (JC-1)
70
Content
JC-1 dye and assay buffer
BD Pharmingen™
BD Pharmingen™ MitoStatus
564696 MitoStatus TMRE
TMRE
BD Pharmingen™ MitoStatus
564697 BD Pharmingen™
© 2016 BD. BD, the BD
Logo and all other trademarks
Company.
MitoStatus
Redare property of Becton, Dickinson and
Red
25mg
100ug
Thank you!
71
張舒婷
新加坡商必帝公司 生物科學部
Product Specialist 產品專員
Mail: [email protected]
Website:
www.bdbiosciences.com/tw
Confidential—For Internal Use Only