Tim Yang
Flow Cytometry Workshop:
Section I:Principles and Concepts
Beckman Coulter Taiwan Branch
技術支援經理
楊人泰
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Basic Principal of Flow Cytometry
5色流式細胞分析儀 Cytomics FC 500
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Watching the Cell: Microscopy
• True Profile of the Cell• Labor-intensive• Arbitrary results
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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)
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讓細胞在管路中排路隊
Hydrodynamic Focusing(流體動力聚焦)
Scatter Signals
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What kinds of light source used in Flow
• Laser (more power but expensive)– 488 nm Blue– 633 nm Red– 405/407/408 nm Violet– 532 nm Green– 325 nm UV or ML UV
• Lamp (cheaper but low sensitivity)
(Could cover >90% applications)
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High angle scatter :Reflection & refraction.Cell structure.
Low angle scatter :Diffraction. Cell size.
Laser
LightDirect beam stop.
Fluorescence signal for specific lableing
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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
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Side Light Scatter:How Granular is that Cell?
Larger Side Scatter
LASERLASER
LASERLASER
Smaller Side Scatter
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Properties of Fluorescent Dyes
Dyes Ex max(nm) Em max(nm) FITC 488 525 PE 488 575 ECD (PE-Tax Red) 488 615 PC5 (PE-Cy5) 488 675 PC7 488 767 PerCP 488 675 PI 488 610 AO 488 530-Green
640-Red
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Flow Cytometry Is Made Of
• Fluid System• Optical System
• Electronic System• Computer System
• Sorting System
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Fluid System: Hydrodynamic Focusing
Flow Cell
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Fluid System: Hydrodynamic Focusing
Sheath
Sample
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• Optical System: Laser & Filter Set
Argon Laser
HeNe Laser
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• Optical System: Filter Set(利用濾片將螢光區分至個別的偵測器中)
FL1
FL2
FL5
FL4
FL3
Side Scatter
600 LP
615 SP
710 LP550 L
P
525 BP
575 BP 755 BP
620
BP
675
BP
500 LP
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• Electronic System: PMT & High Voltage (放大訊號)
Photo-electric Multiplier Tube,光電倍增管
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• Electronic System: Analog to DigitalCell FS SS FL1 FL2
1 543 12 6 25
2
3
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• 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
1 101 50 125 200
2 80 30 700 90
3 20 70 33 234
4 420 26 77 894
OK
OK
╳
╳
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Compensation(設定光補償)
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Compensation(螢光補償)
FL1 PMT:92=90+2 FL2 PMT:100=85+15
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Compensation(螢光補償)
單染FITC:調整 FL2-%FL1
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Compensation(螢光補償)
單染PE:調整 FL1-%FL2
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Compensation(螢光補償)
FITC / PE 雙染
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Flow Raw Data: List Mode Data
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• Computer System: Data Display
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• Computer System: Data Display
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• Computer System: Gate & Analysis
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• Computer System: 多色實驗、多層次Gating
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What’s Sorting ?
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機械式 Sorter vs. 電子式 Sorter
• Sorting Rate: 100~300 cells/sec
• Purity: < 80%
• Cell viability: < 60%
• Time consuming
• Sorting Rate: 15000~30000 cells/sec
• Purity: < 99%
• Cell viability: < 97%
• Time efficient
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Important Issues
Instrument Components: 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)
• Fluid System• Optical System• Electronic System• Computer System• Sorting System
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Data Analysis: Plot & StatisticsData Analysis: Plot & Statistics
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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)
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Three parameter histogram (Tomogram)
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Statistics in Common use
1. Percentage: % Total & %Gate
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2. Mean (MFI): X-mean and Y-mean
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3. CV
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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
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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
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 )
(用以調整 螢光補償)
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Parameter Choice (選擇想要收取的參數)
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Create Plot (利用已選定的參數來做圖)
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Discrimination or Threshold(設定雜訊排除的條件)
FS (size) = 100
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利用Negative Control 調整Voltage and Gain(調整PMT將訊號放大的程度)
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Compensation(設定螢光補償)
單染FITC:調整 FL2-%FL1
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Compensation(設定螢光補償)
單染PE:調整 FL1-%FL2
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利用調整好的設定值正式上檢體
Lymph
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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
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Practice II :DNA Analysis, Cell Cycle
G1
MG2
S G0
Quiescent cells
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DNA Analysis: DNA Content Measurement
DNA Dye:Propidium Iodide (PI)Ethidium BromideAcridine OrangeDAPIHoechst 33342
•• Cell cycle AnalysisCell cycle Analysis
• Ploidy Analysis
• Apoptosis Analysis
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DNA Analysis: Cell Cycle
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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
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Cell cycle + M phase specific protein
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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
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Apoptosis: PI Stain
DNA Content Measurement : Sub-G0
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Apoptosis: AnnexinV + PI
AnnexinV : A phospholipid-binding protein, that has high affinity for PS
PI : A viability dyes that are excluded from viable cells
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Apoptosis: AnnexinV + PI
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Surface Marker:
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Surface Marker:
Background Fewer Binding sites
Larger Number of Binding sites
FL Intensity
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Surface Marker:
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T cell / B cell
NK cell / Suppressor T
Helper T cell
Suppressor T cell
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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
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Cell Function : Calcium kinetic studies
Calcium Tracer:
Indo-1
Fluo-3
Fluo-4
Fluo-5F
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Cell Function : Mitochondria Membrane Potential (Dym)
DioC6: Dym Stainer
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Cell Function : cellular protein content measurements
EGFP & EYFP
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Lyons and Parish, 1994JIM, 171;131
CFSE
Divisions:3 2 1 0
Divisions:3 2 1 0
Using CFSE to track proliferation
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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
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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
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Typical Data
Standard Curves
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Flow analysis of bacteria
Mixed culture (1:1) ofM. 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.
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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:
FL1 FL2 FL3 FL4 FL5
525 nm 575 nm 620 nm 675 nm >755 nm
使用488 雷射 FITC,Alexa488GFP
PE PE-Texas Red, PI
PE-Cy5、PE-Cy5.5、
PerCP、PerCP-Cy5.5、
7-AAD
PE-Cy7、PerCP-Cy7
使用633 雷射 Cy5、APC、
Alexa Fluor 647
APC-Cy7
偵測波長
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3. 外觀及相關部件
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4. 可使用的參數
5. CXP軟體簡易操作說明
Basic Principal of Flow CytometryProperties of Fluorescent DyesCategories of data plotSingle parameter histogramSingle parameter histogramSingle overlayDual parameter histogram (1) --Dot plotThree parameter histogram (Tomogram)