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(1)

Tim Yang

Flow Cytometry Workshop:

Section I:

Principles and Concepts

Beckman Coulter Taiwan Branch 技術支援經理

楊人泰

(2)

Tim Yang

Basic Principal of Flow Cytometry

5色流式細胞分析儀

Cytomics FC 500

(3)

Tim Yang

Watching the Cell: Microscopy

• True Profile of the Cell

• Labor-intensive

• Arbitrary results

(4)

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)

(5)

Tim Yang

讓細胞在管路中排路隊

Hydrodynamic Focusing(流體動力聚焦)

Scatter Signals

(6)

Tim Yang

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)

(7)

Tim Yang

High angle scatter :

Reflection & refraction.

Cell structure.

Low angle scatter : Diffraction. Cell size.

Laser Light

Direct beam stop.

Fluorescence signal for specific lableing

(8)

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

(9)

Tim Yang

Side Light Scatter:How Granular is that Cell?

Larger Side Scatter

LASER LASER

LASER LASER

Smaller Side Scatter

(10)

Tim Yang

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

(11)

Tim Yang

Flow Cytometry Is Made Of

• Fluid System

• Optical System

• Electronic System

• Computer System

• Sorting System

(12)

Tim Yang

Fluid System:

Hydrodynamic Focusing

Flow Cell

(13)

Tim Yang

Fluid System:

Hydrodynamic Focusing

Sheath

Sample

(14)

Tim Yang

• Optical System: Laser & Filter Set

Argon Laser

HeNe Laser

(15)

Tim Yang

• 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 L P

615 S P

710 L 550 L P

P

525 BP

575 BP 755 BP

620 BP675 BP

500 LP

(16)

Tim Yang

• Electronic System: PMT & High Voltage (放大訊號)

Photo-electric Multiplier Tube,光電倍增管

(17)

Tim Yang

• Electronic System: Analog to Digital

Cell FS SS FL1 FL2 1 543 12 6 25 2

3

(18)

Tim Yang

• Electronic System: Leaner Scale Convert to Log Scale

Lin

Log

(19)

Tim Yang

• 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

(20)

Tim Yang

Compensation(設定光補償)

(21)

Tim Yang

Compensation(螢光補償)

FL1 PMT:92=90+2 FL2 PMT:100=85+15

(22)

Tim Yang

Compensation(螢光補償)

單染FITC:調整 FL2-%FL1

(23)

Tim Yang

Compensation(螢光補償)

單染PE:調整 FL1-%FL2

(24)

Tim Yang

Compensation(螢光補償)

FITC / PE 雙染

(25)

Tim Yang

Flow Raw Data: List Mode Data

(26)

Tim Yang

• Computer System: Data Display

(27)

Tim Yang

• Computer System: Data Display

(28)

Tim Yang

• Computer System: Gate & Analysis

(29)

Tim Yang

• Computer System: 多色實驗、多層次Gating

(30)

Tim Yang

What’s Sorting ?

(31)

Tim Yang

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

(32)

Tim Yang

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

(33)

Tim Yang

Data Analysis: Plot & Statistics

Data Analysis: Plot & Statistics

(34)

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)

(35)

Tim Yang

Single parameter histogram

(36)

Tim Yang

Single parameter histogram

(37)

Tim Yang

Single overlay

(38)

Tim Yang

Dual parameter histogram (1) --Dot plot

(39)

Tim Yang

Dual parameter histogram

Counter Plot Density Plot

Surface Plot (Isometric)

(40)

Tim Yang

Three parameter histogram (Tomogram)

(41)

Tim Yang

Statistics in Common use

1. Percentage: % Total & %Gate

(42)

Tim Yang

2. Mean (MFI): X-mean and Y-mean

(43)

Tim Yang

3. CV

(44)

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

(45)

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 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)

(46)

Tim Yang

Parameter Choice (選擇想要收取的參數)

(47)

Tim Yang

Create Plot (利用已選定的參數來做圖)

(48)

Tim Yang

Discrimination or Threshold(設定雜訊排除的條件)

FS (size) = 100

(49)

Tim Yang

利用Negative Control 調整Voltage and Gain

(調整PMT將訊號放大的程度)

(50)

Tim Yang

Compensation(設定螢光補償)

單染FITC:調整 FL2-%FL1

(51)

Tim Yang

Compensation(設定螢光補償)

單染PE:調整 FL1-%FL2

(52)

Tim Yang

利用調整好的設定值正式上檢體

Lymph

(53)

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

(54)

Tim Yang

Practice II :DNA Analysis, Cell Cycle

G1 G2 M

S G0

Quiescent cells

(55)

Tim Yang

(56)

Tim Yang

DNA Analysis:

DNA Content Measurement

DNA Dye:

Propidium Iodide (PI) Ethidium Bromide Acridine Orange DAPI

Hoechst 33342

Cell cycle AnalysisCell cycle Analysis

Ploidy Analysis

Apoptosis Analysis

(57)

Tim Yang

DNA Analysis: Cell Cycle

(58)

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

(59)

Tim Yang

Cell cycle + M phase specific protein

(60)

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

(61)

Tim Yang

Apoptosis: PI Stain

DNA Content Measurement : Sub-G0

(62)

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

(63)

Tim Yang

Apoptosis: AnnexinV + PI

(64)

Tim Yang

Surface Marker:

(65)

Tim Yang

Surface Marker:

Background Fewer Binding sites

Larger Number of Binding sites

FL Intensity

(66)

Tim Yang

Surface Marker:

(67)

Tim Yang

T cell / B cell

NK cell / Suppressor T

Helper T cell

Suppressor T cell

(68)

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

(69)

Tim Yang

Cell Function : Calcium kinetic studies

Calcium Tracer:

Indo-1 Fluo-3 Fluo-4 Fluo-5F

(70)

Tim Yang

Cell Function : Mitochondria Membrane Potential (Dym)

DioC6: Dym Stainer

(71)

Tim Yang

Cell Function : cellular protein content measurements EGFP & EYFP

(72)

Tim Yang

Lyons and Parish, 1994 JIM, 171;131

CFSE

Divisions:

3 2 1 0

Divisions:

3 2 1 0

Using CFSE to track proliferation

(73)

Tim Yang

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

(74)

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

(75)

Tim Yang

Typical Data

Standard Curves

(76)

Tim Yang

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.

(77)

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:

FL1 FL2 FL3 FL4 FL5

525 nm 575 nm 620 nm 675 nm >755 nm

使用488 雷射 FITC, Alexa 488 GFP

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

(78)

Tim Yang

3. 外觀及相關部件

(79)

Tim Yang

4. 可使用的參數

5. CXP軟體簡易操作說明

參考文獻

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