Beckman Coulter Cytomics FC500 Flow Cytometry 自動化五色螢光流式細胞分析儀 Training Course

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 Sheath 488 nm Argon Ion Laser Cross Section sheath sample Flow cell Orifice Waste Sample Flow Cell Single cell suspend (2 10 5 ~10 7 / ml) (12 75 mm tube) Light Scatter Forward Scatter (FS FSC) Side Scatter (SS SSC) Fluorescence (FL) FL1 : 525 nm FL2 : 575 nm FL3 : 620 nm FL4 : 675 nm FL5 : 755 nm Forward Scatter (FS) v.s. Side Scatter (SS) Larger side scatter LASER Larger forward scatter LASER Smaller side scatter Smaller forward scatter Forward scatter size of cells Side scatter granularity of cells Example: Whole blood sample RBC lysed 3 FS Size 1 2 Monos Grans Size: Lymphs Grans > Monos > Lymphs Granularity: SS 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 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 Sample 100 500 events/ sec 4

2. Cytomics FC 500 Optical System: Argon Laser HeNe Laser SSC detector 光電倍增管 (PMT) Flow Cell FSC detector 2. Optical System: Filter Set Side Scatter 500 LP 600 LP 615 SP 620 BP FL3 FL1 525 BP 550 LP 710 LP 675 BP FL4 575 BP 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 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 ) Deflection Plate 調整 : Frequency Drive Amplifier 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 QuickCOMP FITC + PE 雙染實驗 2. 單染 PE 檢體 : 調整 FL1-%FL2 QuickCOMP 14

Compensation ( 螢光補償 ) QuickCOMP ˇ Protocol Setting Flow Setting ( 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 ) 2. 單染 PE: 調整 FL1-%FL2 QuickCOMP 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 FL1 FL2 FL3 FL4 FL5 使用 488 雷射使用 633 雷射 525 nm FITC, Alexa Fluor 488 GFP AO 575 nm PE YFP 620 nm ECD PE-Texas Red, PI AO 675 nm PE-Cy5 PE-Cy5.5 PerCP PerCP-Cy5.5 7-AAD Cy5 APC Alexa Fluor 647 >755 nm PE-Cy7 PerCP-Cy7 APC-Cy7 18

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 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 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 1

Cell Function : Calcium kinetic studies Calcium Tracer: Indo-1 Fluo-3 Fluo-4 Fluo-5F Cell Function : Mitochondria Membrane Potential (Dym) DioC6: Dym Stainer Cell Function : cellular protein content measurements EGFP & EYFP 2

Using CFSE to track proliferation Divisions: 3 2 1 0 Divisions: 3 2 1 0 Lyons and Parish, 1994 JIM, 171;131 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 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 3

Typical Data Standard Curves 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. Cell Surface Marker 4

Surface Markers: 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. Surface Marker: How to identify different surface marker: Using monoclonal antibodies 5

Surface Marker: Background Fewer Binding sites Larger Number of Binding sites FL Intensity Surface Marker: 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 Tube 3: 單染 CD19-PE ( 用以調整螢光補償 ) 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 ) Create Plot ( 利用已選定的參數來做圖 ) Discrimination or Threshold( 設定雜訊排除的條件 ) FS (size) = 100 7

利用 Negative Control 調整 Voltage and Gain ( 調整 PMT 將訊號放大的程度 ) Compensation( 設定螢光補償 ) 單染 FITC: 調整 FL2-%FL1 Compensation( 設定螢光補償 ) 單染 PE: 調整 FL1-%FL2 8

利用調整好的設定值正式上檢體 Lymph 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 ) 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) 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??? 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/ Critical Issues: 2. Dye Choice D. Dye Sensitivity: 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 uncompensated partially compensated fully compensated FL2 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 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 DNA content Analysis (Cell cycle & Apoptosis) 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 Application 1: cell cycle analysis Cells contain different DNA content through cell cycle 4C 4C G2 M 2C 2C~4C S G1 2C G0 Quiescent cells Application 1: cell cycle analysis 12

Application 1: cell cycle analysis 4C 4C G2 M 2C Stained with PI, analyzed by Flow 2C~4C S 2C G1 G0 Cell Numbers 2C 2C~4C 4C DNA content or FL intensity Application 1: cell cycle analysis G 0 -G 1 % G 2 -M % # of Events S % Fluorescence Intensity Application 1: cell cycle analysis Cell cycle (PI stained) + mataphase specific protein (phospho-histon-h3) 13

Application 1: cell cycle analysis BromodeoxyUridine (BrdU) Incorporation Control (left) versus drug treated (right) effect on cell cycle 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 Application 3: DNA index analysis 14

Application 4: apoptosis (sub-g0/g1) analysis DNA Content Measurement : Sub-G0/G1 Control Drug treated 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 FS FL2 or FL3 How to gate out the doublet cell before FL plot display?? The signaling process by Flow A B C There are 3 kinds of signals could be measured (H) (A) 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 15

To measure the signals between singlet and doublet : Peak (H) Integral (A) 1 2 1 1 2 2 TOF (W) 1 1 2 Doublet discrimination: method 1, using H and A Peak (H) Integral (A) 1 2 1 1 2 2 Doublet discrimination: method 1, using H and A You could use this method on XL, FC500, FACSCanto, Cyan. 16

Doublet discrimination: method 2, using H, A and H/A Peak (H) Integral (A) 1 2 1 1 2 2 Ratio (H/A) 1 1 ½ or <1 Doublet discrimination: method 2, using H, A and H/A You could use this method on XL, FC500.. Doublet discrimination: method 3, using A and W Integral (A) Tim of Flight (W) 1 2 2 1 1 2 17

Doublet discrimination: method 3, using A and W You could use this method on FACScan, FACSCalibur, FACSCanto. So the real process are. SS FS FL2 or FL3 Cell cycle analysis: real practice on FC500 1. Parameters selection 18

2. Plots Creation 3. Instrument Setup: Run Control Cell Staining with PI: 調整 FS SS FL3 Lin(Int) FL3 Peak Voltage & Gain Final Result Other critical issues: 1. The proficiency of sample preparation: the CV of G0G1 phase Good resolution Bad resolution Solution: 19

Other critical issues: 2. How to resolve GoG1 / S / G2M phase? G 0 -G 1 % Manually G 2 -M % By Software: Phoenix MultiCycle or Verity Mofit # of Events S % 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/ 20