Beckman Coulter Cytomics FC500 Flow Cytometry

Beckman Coulter Cytomics FC500 Flow Cytometry

Beckman Coulter
Cytomics FC500 Flow Cytometry
自動化五色螢光流式細胞分析儀
Training Course
美商貝克曼庫爾特公司台灣分公司
http://www.beckman.com
Principle and Concept of
Cytomics FC500 Flow Cytometer
產品專員 李明純 Sabrina Lee
美商貝克曼庫爾特公司台灣分公司
生物醫學部 0800211283
Watching the Cell: Microscopy
• True Profile of the Cell
• Labor-intensive
• Arbitrary results
Why use Flow Cytometry
• More fast, >100 events per second date rate
• 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
• Analyze with multi-parameters of cell information
That’s Why we use Flow Cytometry, FCM, or
FACS (Fluorescence Activated Cell Sorter)
1
Basics of Flow cytometry
Sample
Single cell suspend
(2×105～107 / mL)
(12 × 75 mm tube)
Flow Cell
Light Scatter
Sheath
Forward Scatter (FS、FSC)
Side Scatter (SS、SSC)
488 nm Argon
Ion Laser
Fluorescence (FL)
FL1 : 525 nm
FL2 : 575 nm
Cross Section
FL3 : 620 nm
sample
FL4 : 675 nm
Waste
Flow cell
Orifice
sheath
FL5 : 755 nm
Forward Scatter (FS) v.s. Side Scatter (SS)
Larger side scatter
Larger
forward
scatter
LASER
Smaller side scatter
Smaller
forward
scatter
LASER
Forward scatter ∝ size of cells
Side scatter ∝ granularity of cells
Example: Whole blood sample RBC lysed
FS Size
3
2
Grans
1
Monos
Lymphs
SS granularity
Size:
Grans > Monos > Lymphs
Granularity:
Grans > Monos > Lymphs
2
What kinds of light source used in Flow
• Laser (more power but expensive)
– 488 nm Blue (Could cover > 90% applications)
– 633 nm Red (For APC dye)
– 405/407/408 nm Violet
– 532 nm Green
– 325 nm UV or ML UV (For DAPI, Hoechst 33342)
Common Fluorochromes used in Flow
FL1
FL2 FL3 FL4
FL5
ˇ
ˇ
3
Flow Cytometry Is Made Of
1. Fluid System
2. Optical System
3. Electronic System
4. Computer System
5. Sorting System
自動化五色流式細胞分析儀
自動化五色流式細胞分析儀
1. Fluid System: Hydrodynamic Focusing (流體動力聚焦)
Flow Cell
1. Fluid System: Hydrodynamic Focusing (流體動力聚焦)
Sheath
100 – 500 events/ sec
Sample
4
2. Cytomics FC 500 Optical System:
HeNe Laser
Argon Laser
SSC detector
光電倍增管(PMT)
Flow Cell
FSC detector
2. Optical System: Filter Set
SP
LP
55
0
0
71
LP
LP
525 BP
575 BP
620 BP
5
61
0
60
500 LP
FL1
FL3
675 BP
Side
Scatter
FL4
755 BP
FL5
FL2
• BandPass (BP)
• Dichroic LongPass (DL)
• Dichroic ShortPass (DS)
3. Electronic System: 光電倍增管 (Photomultipliers, PMT)
& 放大訊號(High Voltage)
5
3. Electronic System: Discrimination or Threshold (排除雜訊)
FS ＜ 100
FS ＞ 100
FS = 60 ～ 100
3.
Electronic System: 將線性訊號轉換成Log訊號
FS、
FS、SS
Lin
FL1 – FL5
Log
Parameter Selection: Lin & Log signal
6
Flow Raw Data
4. Computer System: Data Display
Dot Plot
4. Computer System: Data Display
Histogram Plot
7
4. Computer System: Gating & Analysis
4. Computer System : 多色實驗、多層次Gating
5. Sorting System: Electronic Sorting (電子式細胞分選系統)
調整：
Frequency
Drive
Amplifier
Deflection Plate
設定三條水柱
計算 Time Delay
• Sorting Rate: up to 70,000 cells/sec
• Purity: > 99% at all speed
• Cell recovery: > 95%
• Cell viability: > 95%
• Time efficient
8
5. Sorting System: Result
Important Issues
Instrument Components:
•
•
•
•
•
Fluid System
Optical System
Electronic System
Computer System
Sorting System
Concepts:
•
•
•
•
•
•
•
•
Hydrodynamics Focusing
Filter Set
PMT & High Voltage
Lin & Log Data
Discriminator or Threshold
List Mode Data
Gating and Analysis
Sorting
Take a Break !!
9
Data Analysis: Plot & Statistics
Plots
1. Single parameter histogram
1. Single parameter histogram overlap
10
2. Dual parameter histogram – (1) Dot plot
2. Dual parameter histogram – (2) Contour plot
(等高線圖)
2. Dual parameter histogram – (3) Density plot
11
2. Dual parameter histogram – (4) Isometric plot
( Surface plot )
3. Three parameter histogram (Tomogram)
Statistics
1. Percentage : % Total & %Gate
12
2. Mean : X-mean and Y-mean
3. Coefficient of Variant : CV (變異係數)
Compensation (螢光補償)
525
575
620
675
755
FL1
FL2
FL3
FL4
FL5
13
FITC + PE 雙染實驗
FL1 PMT：92＝90+2
FL2 PMT：100＝85+15
FITC + PE 雙染實驗
1. 單染FITC檢體：調整 FL2-%FL1
FITC + PE 雙染實驗
2. 單染PE檢體：調整 FL1-%FL2
QuickCOMP
QuickCOMP
14
Compensation (螢光補償)
QuickCOMP
ˇ
Protocol Setting
實驗的必要步驟)
)
Flow Setting (設定一個Flow
設定一個Flow實驗的必要步驟
™ 上樣前：
1.
Choice Parameter (Lin or Log)
2.
Create Displaying Plot
3. Setting Discrimination
™ 上檢體：
4.
利用Control cells 調整偵測器訊號放大程度
(Voltage and Gain)
5.
利用單染檢體調整螢光補償值
(Compensation)
™ All of the setting save in the Protocol
™ 利用調整好的條件正式上檢體
15
1. Parameter Choice （選擇想要收取的參數）
2. Create Plot （利用已選定的參數來做圖）
3. Discrimination（設定雜訊排除的條件）
FS (size) = 60 - 100
16
4. 利用Negative Control 調整Voltage and Gain
QuickSET
5. 利用單染檢體調整 Compensation (螢光補償)
525
575
620
675
755
Compensation（螢光補償）
QuickCOMP
1. 單染FITC：調整 FL2-%FL1
17
Compensation（螢光補償）
QuickCOMP
2. 單染PE：調整 FL1-%FL2
™ 利用調整好的條件正式上檢體
Lymph
Beckman Coulter Cytomics 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
FL1
FL2
FL3
FL4
FL5
525 nm
575 nm
620 nm
675 nm
>755 nm
FITC,
Alexa Fluor
488
GFP
AO
PE
YFP
ECD
PE-Texas Red,
PI
AO
PE-Cy5、
PE-Cy5.5、
PerCP、
PerCP-Cy5.5、
7-AAD
PE-Cy7、
PerCP-Cy7
Cy5、
APC、
Alexa Fluor 647
APC-Cy7
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3. 外觀及相關部件
4. 可使用的參數
5.
CXP Software
CXP Cytometer：儀器操控軟體，可與機器連線，收取List Mode Data，進
行簡單的數據分析。
CXP Analysis：離線分析軟體，可進行較繁複的數據分析，疊圖繪製，多檔
案分析與批次分析。
Thanks for Your Attention !!
19
Flow Cytometry Lesson：
Section II:
Current Applications of Flow
Beckman Coulter Taiwan Branch
技術支援經理
楊人泰
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
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
1
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
2
Using CFSE to track proliferation
Divisions:
3 2 1 0
Divisions:
3
2
1
0
Lyons and Parish, 1994
JIM, 171;131
CFSE
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
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
3
Typical Data
Standard Curves
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.
Tim Yang
Cell Surface Marker
Tim Yang
4
Surface Markers:
Tim Yang
Surface Markers:
• Surface molecular is useful to identify cell type, differential stage and cell function.
• Researchers used different names for these molecular.
• CD (Cluster designation) numbers are assigned from International workshop on
Human Leucocyte Differentiation Antigens (HLDA)
• The first workshop was held in 1982.
• HLDA-8 was held in 2005 and assigned CD number to CD 339.
Tim Yang
Surface Marker:
How to identify different surface marker: Using monoclonal antibodies
Tim Yang
5
Surface Marker:
Background
Fewer Binding
sites
Larger Number of
Binding sites
FL Intensity
Tim Yang
Surface Marker:
Tim Yang
範例一：Double stain surface marker
WBC 以 CD3-FITC （T 細胞）,
CD19-PE （B 細胞）染色
概念：先在 FS /SS Dot Plot上抓到淋巴球，再以FL1 Log/FL2 Log
Dot Plot 觀察這兩種 Marker 的染色狀況
必須收取的參數：FS Lin, SS Lin, FL1 Log, FL2 Log
調整機器必須準備的檢體：
• Tube 1: Negative control (unstain or isotypic stain)（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
Tim Yang
• Tube 4：雙染 CD3-FIT / CD19-PE （用以確認條件的正確與否 ）
6
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
7
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
8
利用調整好的設定值正式上檢體
Lymph
Tim Yang
範例二：5 colors surface marker （FITC / PE / ECD / PC5 / PC7）
1.
需要的參數( FS Lin, SS Lin, FL1 ~5 Log)
2.
調整機器需準備的檢體：
• Tube 1: Negative control (unstain or isotypic stain)（用以調整 FS,
SS, FL1~FL5 的電壓值）
• Tube 2：單染 FITC
• Tube 3：單染 PE
• Tube 4：單染 ECD
（用以調整螢光補償）
• Tube 5：單染 PC5
• Tube 6：單染 PC7
• Tube 7：5 種螢光色同時染色（用以確認條件的正確與否 ）
Tim Yang
Critical Issues： 1. Negative Control: Blank (unstain) vs. Isotypic Control
Un-Stain
Auto-Fluorescence only(0)
Isotypic Control
Auto Fluorescence(0) +
Ab non-Specific Binding(3)
Positive Cell
Auto Fluorescence(0) +
non-Specific Binding(3) +
Specific Binding(99)
Tim Yang
9
Critical Issues： 1. Negative Control: Blank (unstain) vs. Isotypic Control
• non-specific binding 取決於抗體的種類 （Isotype）
• 如何選擇 Isotypic Control
• 能否不要使用 Isotypic stain 最為 Negative Control ？？？
Tim Yang
Critical Issues： 2. Dye Choice
A. Light Source and Emission Channel
B. Fluorochromes Excited by different Laser:
488nm：FITC, Alexa 488, PE, PE-Texas Red, PE-Cy5, PE-Cy7
633/536 nm：Cy5, APC, Alexa 647, Alexa 700, Alexa 750, APC-Cy7
405/407/408 nm：Alexa 405, Pacific Blue, Pacific Orange
Invetrogen Fluorescence Spectra Viewer:
http://probes.invitrogen.com/resources/spectraviewer/
Tim Yang
Critical Issues： 2. Dye Choice
D. Dye Sensitivity：
Tim Yang
CD8-FITC
CD8-PE
CD8-ECD
CD8-PC5
CD8-PerCP
CD8-APC
PerCP < APC < FITC < ECD < PC5 < PE
10
Critical Issues： 3. Manually vs. Auto Compensation
partially
compensated
fully
compensated
FL2
uncompensated
FL1
• Manual Compensation 使用肉眼判斷螢光補償值是否合適
• 通常可使用平均值或中位數輔助判斷
• 當進行多色實驗時，因染劑光譜重疊複雜，常不易執行人工螢光補償
Tim Yang
Critical Issues： 3. Manually vs. Auto Compensation
• 在沒有設定任何螢光補償的情況下，量測每種染劑進入個別螢光通道的強度
• 軟體綜合量測結果，計算出完整的 Compensation Matrix
Tim Yang
DNA content Analysis
(Cell cycle & Apoptosis)
Tim Yang
11
DNA Content Analysis-common sense
• DNA content analysis is one of the oldest Flow applications.
• Using appropriate dye to label DNA and then measuring the
fluorescence by flow cytometry
• DNA dyes using on flow cytometry:
- Propidium Iodide (PI)
- DAPI
- 7-AAD
- Hoechst 33342, 33258
- Acridine Orange (AO) - Vybrant DyeCycle
• Familiar applications using this technology:
- Cell cycle analysis
- Ploidy analysis
- DNA index analysis
- Apoptosis sub-G1 analysis
Tim Yang
Application 1: cell cycle analysis
Cells contain different DNA content through cell cycle
4C
4C
G2
M
2C
G1
2C~4C
S
2C
G0
Quiescent cells
Tim Yang
Application 1: cell cycle analysis
Tim Yang
12
Application 1: cell cycle analysis
4C
4C
G2 M
G1
S
2C
G0
Cell Numbers
2C~4C
Stained with PI,
analyzed by Flow
2C
2C
4C
2C~4C
Tim Yang
DNA content or FL intensity
Application 1: cell cycle analysis
G2-M
%
# of Events
G0-G1
%
Tim Yang
S
%
Fluorescence Intensity
Application 1: cell cycle analysis
Cell cycle (PI stained) + mataphase specific protein (phospho-histon-H3)
Tim Yang
13
Application 1: cell cycle analysis
BromodeoxyUridine (BrdU) Incorporation
Control (left) versus drug treated (right) effect on cell cycle
Tim Yang
Application 2: 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
Application 3: DNA index analysis
Tim Yang
14
Application 4: apoptosis (sub-G0/G1) analysis
DNA Content Measurement : Sub-G0/G1
Control
Drug treated
Tim Yang
The process: cell stained with PI, gating the major population from
SS/FS plot and display gated cell on fluorescent (FL2
or FL3) histogram
But………PI usually bring the doublet problem……..
Doublet Problem
SS
?
FL2 or FL3
FS
How to gate out the doublet cell before FL plot display??
Tim Yang
The signaling process by Flow
There are 3 kinds of signals could be
measured
(H)
A
(A)
B
C
Time of Flight (W)
In FACSan and FACSCalibur, we used to used ‘H’ as the major parameter
In FC500, XL, and FACSCanto, we used to used ‘A’ as the major parameter
Tim Yang
15
To measure the signals between singlet and doublet :
Peak (H)
1
2
1
Integral (A)
1
2
2
TOF (W)
1
1
2
Tim Yang
Doublet discrimination: method 1, using H and A
Peak (H)
1
2
1
Integral (A)
1
2
2
Tim Yang
Doublet discrimination: method 1, using H and A
You could use this method on XL, FC500, FACSCanto, Cyan…….
Tim Yang
16
Doublet discrimination: method 2, using H, A and H/A
Peak (H)
1
2
1
Integral (A)
1
2
2
Ratio (H/A)
1
1
½ or <1
Tim Yang
Doublet discrimination: method 2, using H, A and H/A
You could use this method on XL, FC500……..
Tim Yang
Doublet discrimination: method 3, using A and W
Integral (A)
1
2
2
Tim of Flight (W)
1
1
2
Tim Yang
17
Doublet discrimination: method 3, using A and W
You could use this method on FACScan, FACSCalibur, FACSCanto…….
Tim Yang
SS
So… the real process are……………….
FS
FL2 or FL3
Tim Yang
Cell cycle analysis: real practice on FC500
1. Parameters selection
Tim Yang
18
2. Plots Creation
3. Instrument Setup:
Run Control Cell Staining with PI:
調整 FS、SS、FL3 Lin(Int)、FL3 Peak Voltage & Gain
Tim Yang
Final Result
Tim Yang
Other critical issues:
1. The proficiency of sample preparation: the CV of
G0G1 phase
Good resolution
Bad resolution
Solution:
Tim Yang
19
Other critical issues:
2. How to resolve GoG1 / S / G2M phase?
Manually
G2-M %
# of Events
G0-G1 %
By Software:
Phoenix MultiCycle or
Verity Mofit
S%
Tim Yang
Fluorescence Intensity
Some Flow relative web-sites:
Purdue University Cytometry Laboratories
http://www.cyto.purdue.edu/
Purdue University Cytometry Laboratories‘ E-mail Archive
http://www.cyto.purdue.edu/hmarchiv/cytomail.htm
陽明大學儀器中心及 Flow 討論區
http://140.129.64.205/Xoops224/modules/sign_up/
NCI ETI Branch Flow Cytometry Core Laboratory
http://home.ncifcrf.gov/ccr/flowcore/index.htm
Invitrogen’s Fluorescence Spectra Viewer
http://probes.invitrogen.com/resources/spectraviewer/
Tim Yang
20