Flow Cytometry Workshop： Section I: Principles and Concepts

Flow Cytometry Workshop： Section I: Principles and Concepts

Flow Cytometry Workshop：
Section I:
Principles and Concepts
Beckman Coulter Taiwan Branch
技術支援經理
楊人泰
Tim Yang
Basic Principal of Flow Cytometry
5色流式細胞分析儀 Cytomics FC 500
Tim Yang
Watching the Cell: Microscopy
• True Profile of the Cell
• Labor-intensive
• Arbitrary results
Tim Yang
Can we design an instrument to see cells…….
• more fast, >100 events per second
• with the capability to acquire large amount of cell
numbers to improve the accuracy of statistics.
• more objectively between you and me
• more sensitively than eyes
• still with multi-parameters of cell information
That’s Why we use Flow Cytometry, FCM,
or FACS (Fluorescence Activated Cell Sorter)
Tim Yang
讓細胞在管路中排路隊
Hydrodynamic Focusing（流體動力聚焦）
Scatter Signals
Tim Yang
What kinds of light source used in Flow
• Laser (more power but expensive)
–
–
–
–
–
488 nm Blue (Could cover >90% applications)
633 nm Red
405/407/408 nm Violet
532 nm Green
325 nm UV or ML UV
• Lamp (cheaper but low sensitivity)
Tim Yang
High angle scatter :
Reflection & refraction.
Cell structure.
Low angle scatter :
Diffraction. Cell size.
Laser
Direct beam stop.
Light
Fluorescence signal for
specific lableing
Tim Yang
What Flow Cytometry Give You
• Light Scatter for cell morphology
– Forward Angle Light Scatter (FS) for cell size
– Side Scatter (SS) for cell complexity
• Fluorescenses (FL) for specific labeling
–
–
–
–
–
FL 1 : (525 nm BP) for green dyes
FL 2 : (575 nm BP) for yellow dyes
FL 3 : (615 nm BP) for orange dyes
FL 4 : (675 nm BP) for red dyes
FL 5 : (>750 nm BP) for deep red dyes
Tim Yang
Side Light Scatter：How Granular is that Cell?
Larger Side Scatter
LASER
Smaller Side Scatter
LASER
Tim Yang
Properties of Fluorescent Dyes
Tim Yang
Dyes
Ex max(nm)
Em max(nm)
FITC
PE
ECD (PE-Tax Red)
PC5 (PE-Cy5)
PC7
PerCP
PI
AO
488
488
488
488
488
488
488
488
525
575
615
675
767
675
610
530-Green
640-Red
Flow Cytometry Is Made Of
• Fluid System
• Optical System
• Electronic System
• Computer System
• Sorting System
Tim Yang
Fluid System: Hydrodynamic Focusing
Tim Yang
Flow Cell
Fluid System: Hydrodynamic Focusing
Sheath
Sample
Tim Yang
• Optical System: Laser & Filter Set
HeNe Laser
Argon Laser
Tim Yang
• Optical System: Filter Set（利用濾片將螢光區分至個別的偵測器中）
LP
LP
55
0
0
71
755 BP
620 BP
SP
LP
525 BP
575 BP
FL5
FL2
Tim Yang
5
61
0
60
500 LP
FL1
FL3
675 BP
Side
Scatter
FL4
• Electronic System: PMT & High Voltage （放大訊號）
Photo-electric Multiplier Tube,光電倍增管
Tim Yang
• Electronic System: Analog to Digital
Cell
FS
SS
1
543
12
2
3
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FL1 FL2
6
25
• Electronic System: Leaner Scale Convert to Log Scale
Lin
Log
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• Electronic System: Discrimination or Threshold（排除雜訊）
Example:
Discriminator: FS=100
Cell
FS SS FL1 FL2
125 200
OK
1
101 50
2
80 30
700
90
╳
3
20 70
33
234
╳
4 Yang
420 26
Tim
77
894
OK
Compensation（設定光補償）
Tim Yang
Compensation（螢光補償）
FL1 PMT：92＝90+2
Tim Yang
FL2 PMT：100＝85+15
Compensation（螢光補償）
單染FITC：調整 FL2-%FL1
Tim Yang
Compensation（螢光補償）
單染PE：調整 FL1-%FL2
Tim Yang
Compensation（螢光補償）
FITC / PE 雙染
Tim Yang
Flow Raw Data: List Mode Data
Tim Yang
• Computer System: Data Display
Tim Yang
• Computer System: Data Display
Tim Yang
• Computer System: Gate & Analysis
Tim Yang
• Computer System: 多色實驗、多層次Gating
Tim Yang
What’s Sorting ?
Tim Yang
機械式 Sorter vs. 電子式 Sorter
• Sorting Rate: 100~300 cells/sec
• Sorting Rate: 15000~30000 cells/sec
• Purity: < 80%
• Purity: < 99%
• Cell viability: < 60%
• Cell viability: < 97%
• Time consuming
• Time efficient
Tim Yang
Important Issues
Instrument Components:
•
•
•
•
•
Fluid System
Optical System
Electronic System
Computer System
Sorting System
Tim Yang
Concepts:
•
•
•
•
•
•
•
•
•
Hydrodynimics Focusing
Filter Set
PMT & High Voltage
Lin Data & Log Data
Discriminator or Threshold
Color Compensation
List Mode Data
Gating and Analysis
Sorting (電子式、機械式)
Data Analysis: Plot & Statistics
Tim Yang
Categories of data plot
• Single parameter histogram
• Dual parameter histogram
– Dot plot
– Contour plot
– Density plot
– Isometric plot (+ Count)
• Three parameter histogram(Tomogram)
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Single parameter histogram
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Single parameter histogram
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Single overlay
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Dual parameter histogram (1) --Dot plot
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Dual parameter histogram
Counter Plot
Density Plot
Surface Plot (Isometric)
Tim Yang
Three parameter histogram (Tomogram)
Tim Yang
Statistics in Common use
1. Percentage: % Total & %Gate
Tim Yang
2. Mean (MFI): X-mean and Y-mean
Tim Yang
3. CV
Tim Yang
Flow Setting （設定一個Flow實驗的必要步驟）
™上樣前：
• Choice Parameter
• Create Displaying Plot
• Setting Discrimination (Threshold)
™上檢體：
• 利用 N.C. 調整每個參數的Voltage, Gain
• 利用單染檢體調整Compensation
™All of The Setting Save in the Protocol
™利用調整好的條件正式上檢體
Tim Yang
範例：Double stain surface marker
WBC 以 CD3-FITC （T 細胞）,
CD19-PE （B 細胞）染色
概念：先在 FS/SS 的圖上抓到淋巴球，再以FL1/FL2雙參數圖觀察
這兩種 Marker 的染色狀況
必須收取的參數：FS, SS, FL1 Log, FL2 Log
調整機器必須準備的檢體：
Tube 1: Unstain cell or isotype（用以調整 FS, SS, FL1, FL2 的電壓
值）
Tube 2：單染CD3-FITC
（用以調整 螢光補償）
Tube 3：單染CD19-PE
Tube 4：雙染 CD3-FIT / CD19-PE （用以確認條件的正確與否 ）
Tim Yang
Parameter Choice （選擇想要收取的參數）
Tim Yang
Create Plot （利用已選定的參數來做圖）
Tim Yang
Discrimination or Threshold（設定雜訊排除的條件）
FS (size) = 100
Tim Yang
利用Negative Control 調整Voltage and Gain
（調整PMT將訊號放大的程度）
Tim Yang
Compensation（設定螢光補償）
單染FITC：調整 FL2-%FL1
Tim Yang
Compensation（設定螢光補償）
單染PE：調整 FL1-%FL2
Tim Yang
利用調整好的設定值正式上檢體
Lymph
Tim Yang
Current Applications of Flow Cytometry:
• DNA analysis: cell cycle analysis,
tumor monitoring,
detection of ploidy,
• Understanding Apoptosis:
• Cell- surface marker immunofluoresecnce analysis:
Stem cells tracking and enumeration;
Analysis of platelets;
Leukocyte Immunophenotyping;
HIV infection
Tim Yang
Practice II :DNA Analysis, Cell Cycle
G2
M
G1
S
G0
Quiescent cells
Tim Yang
Tim Yang
DNA Analysis: DNA Content Measurement
DNA Dye:
Propidium Iodide (PI)
Ethidium Bromide
Acridine Orange
DAPI
Hoechst 33342
• Cell cycle Analysis
Tim Yang
•
Ploidy Analysis
•
Apoptosis Analysis
DNA Analysis: Cell Cycle
Tim Yang
DNA Analysis: Ploidy Analysis
•
•
•
Hyperdiploid: greater than the normal 2n number of chromosomes
Hypodiploid: Less than the normal 2n number of chromosomes
Tetraploidy: Containing double the number of chromosomes
Tim Yang
Cell cycle + M phase specific protein
Tim Yang
Apoptosis:
Programmed cell death or apoptosis is the most common form of eukaryotic
cell death. It is a physiological suicide mechanism that preserves homeostasis,
in which cell death naturally occurs during normal tissue turnover.
* PS (Phosphatidylserine)
Translocation
* DNA Fragmentation
Tim Yang
Apoptosis: PI Stain
DNA Content Measurement : Sub-G0
Tim Yang
Apoptosis: AnnexinV + PI
AnnexinV : A phospholipid-binding protein, that has high affinity for PS
PI : A viability dyes that are excluded from viable cells
Tim Yang
Apoptosis: AnnexinV + PI
Tim Yang
Surface Marker:
Tim Yang
Surface Marker:
Background
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Fewer Binding
sites
FL Intensity
Larger Number of
Binding sites
Surface Marker:
Tim Yang
T cell / B cell
Helper T cell
NK cell / Suppressor T
Suppressor T cell
Tim Yang
Applications of Flow Cytometry: Overview
• Cell function assays:
• calcium kinetic studies：Ca++ Con., Ca++ kinetic
• mitochondria membrane potential：DioC6, JC1
• cellular protein content measurements：GFP, YFP….
• cell proliferation：CFSE
• analysis of intracellular proteins：intracellular cytokine
• cellular RNA and DNA content：reticulocyte, SCSA
• intracellular pH, proton pump activity
Tim Yang
Cell Function : Calcium kinetic studies
Calcium Tracer:
Indo-1
Fluo-3
Fluo-4
Fluo-5F
Tim Yang
Cell Function : Mitochondria Membrane Potential (Dym)
DioC6: Dym Stainer
Tim Yang
Cell Function : cellular protein content measurements
EGFP & EYFP
Tim Yang
Using CFSE to track proliferation
Divisions:
3 2 1 0
Divisions:
3
2
1
0
Lyons and Parish, 1994
JIM, 171;131
Tim Yang
CFSE
Applications of Flow Cytometry:
• Detection of soluble protein： beads based multiplex assay
• Enzyme kinetics
• Detection of intracellular virus and viral products
• Environmental microrganism detection, aquatic-organism
studies
• Chromosomes analysis and sorting
• Sex specific sperm sorting
Tim Yang
Beads-based multiplexed cytokine assay (Multiplex ELISA)
1. 置備檢體
2. 上機及收取數據
3. 利用自動化分析
軟體繪製標準曲
線，並計算未知
檢體的濃度
Tim Yang
Typical Data
Standard Curves
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Flow analysis of bacteria
Mixed culture (1:1) of
M. luteus (gram-positive cocci)
and E. coli (gram-negative rods)
Isometric flow-cytometric plots. (A) E. coli; (B) M. luteus; (C) mixed culture (1:1) of E. coli and M. luteus.
Tim Yang
Beckman Coulter FC500 簡介
1.
配備488nm (藍色) 及633nm (紅色) 兩種雷射激發波長。
2.
可同時偵測 5 種螢光訊號，偵測範圍及相關染劑如下表：
偵測波長
使用488 雷射
使用633 雷射
Tim Yang
FL1
FL2
FL3
FL4
FL5
525 nm
575 nm
620 nm
675 nm
>755 nm
FITC,
Alexa
488
GFP
PE
PE-Texas Red,
PI
PE-Cy5、
PE-Cy5.5、
PerCP、
PerCP-Cy5.5、
7-AAD
PE-Cy7、
PerCP-Cy7
Cy5、
APC、
Alexa Fluor 647
APC-Cy7
3. 外觀及相關部件
Tim Yang
4. 可使用的參數
5. CXP軟體簡易操作說明
Tim Yang