flow cytometry workshop section i: principles and concepts · principles and concepts beckman...

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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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單染FITC：調整 FL2-%FL1

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單染PE：調整 FL1-%FL2

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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: 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 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)

flow cytometry workshop section i: principles and concepts · principles and concepts beckman...

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