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Flow Cytometry Basic Introduction and Principle Cloe Chang 張舒婷 Product Specialist BD Bioscience [email protected] Sep 12, 2016 © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 1 Topic Introduction of Flow Cytometry Instrument setup Application of Flow Cytometry 2 © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. What is Flow Cytometry? Flow = Fluid Cyto = Cell Metry = Measurement A variety of measurements are made on cells, cell organelles, and other objects suspended in a liquid and flowing at rates of several thousands per second through a flow chamber. Confidential—For Internal Use Only Particle Size • Detection range: 0.5~50m © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. What Can a Flow Cytometer Tell Us About a Cell? • Its relative size (Forward Scatter—FSC;前向散射光) • Its relative granularity or internal complexity (Side Scatter—SSC;側向散射光) • Its relative fluorescence intensity © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Scatter Light Forward Laser FSC Sensor o 90 SSC Sensor © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Side Scatter (SSC) Ex. Lysed Whole Blood Neutrophils Monocytes Forward Scatter (FSC) Lymphocytes © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Fluorescence Light © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Fluorescence intensity © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. BD Flow Cytometers – Cell Analyzer FACSCalibur FACSCanto 2 Lasers, 4 Colors Analyzer 2 Lasers, 6 Colors Analyzer © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. BD Flow Cytometers – Cell Sorter FACSAria 3 Lasers, 11 Colors Sorter © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Main Component Fluidics 液流系統 To introduce and focus the cells for interrogation. Optics 光學系統 To generate and collect the light signals. Electronics 電子系統 To convert the optical signals to proportional digital signals, process the signals, and communicate with the computer. © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Fluidics Pressurized System © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Sample Flow Excitation Lasers Hydrodynamic Focusing Flow Cell sheath sheath sample © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Sample Differential © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Optical Excitation optics: Lasers: 488nm(Blue) + 633nm(Red) Filters and mirrors that route the laser light to the fluid stream Collection optic: Fiber optic cables that direct the emitted light to the appropriate emission block Filters that direct the signals in the emission block to the appropriate photomultiplier tube (PMT) © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Excitation and Emission Use the maximum excitation wavelengths to determine lasers that can be used to excite the fluorochrome. Use the maximum emission wavelengths to determine filters and PMTs that can be used to measure the signal. 488 530/30 Emission Excitation FITC © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. FACSCalibur Optics Collection optics Excitation optics © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Optical Filters © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. FACSCalibur Optics FL1 530/30 SSC FL2 FL4 Collection Optics 488/10 585/42 90/10 Beam Splitter 661/16 DM 560SP DM 640LP 670LP Half Mirror Excitation Optics 488 nm Blue Laser Fluorescence Collection Lens FL3 Flow Cell ~635 nm Red Diode Laser 488/10 Focusing Lens © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. FSC Diode FACSCalibur Configuration 488nm 635nm © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. FACSCanto Excitation Optics 633nm 488nm © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. FACSCanto Collection Optics © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Collection Optics—Octagon Blue Laser Signal • 接收光源順序:能量弱→強 750–810 nm • 反射傳遞:能量折損較低 564–606 nm 515–545 nm 483–493 nm > 670 nm © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Collection Optics—Trigon Red Laser Signal 650–670 nm 750–810 nm © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. FACSCanto —Octagon and Trigons (BD專利設計) 因為專利光學設計,使FACSCanto 流式細胞儀靈敏度及解析度皆提升! © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. BD FACSCanto Configuration © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Electronics PMTs and preamps convert photons to voltage pulses. Analog-to-digital converters translate analog signals to proportional digital signals. Compute area and height for each pulse. Perform compensation and calculate ratios and width. An embedded computer interfaces with the computer workstation for data transfer. © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Pulse Height Laser Volts Creation of a Voltage Pulse Time (µs) Pulse Width © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Quantification of a Voltage Pulse © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Data Storage Histogram List-Mode Data Time FSC SSC FITC PE Event 1 0 60 120 675 39,271 39,271 89 Event 2 10 160 65 39,271 30,621 Event 3 30 650 160 22,688 6,189 Dot Plot FITC FITC 39,271 89 675 PE 30,621 © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. PE Data Display Linear Scaling Log Scaling © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Linear vs. Log Volts FL1 Voltage Pulses Linear mode 0.1 V Volts Time 0.4 V Pulse Height 0V 10 V Volts Time 1.2 V Log mode Volts Time 4V Volts Time 8V 0.001 V Time © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 0.01 V 0.1 V Pulse Height 1V 10 V Linear v. Log Amplification Linear amplification is usually used for light scatter parameters and DNA analysis. Log amplification is used for fluorescence signals with a large dynamic range. © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Flow Cytometry Detection Principle Time SSC 1. fluidics Time FSC Time PE Data Processing FITC APC Time Time 2. optics PerCP-Cy5.5 Time Computer operation 3. electronics © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Topic Introduction of Flow Cytometry Instrument setup Application of Flow Cytometry 36 © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Auto-fluorescence Non-stain sample UL UR LL LR Auto-fluorescence After voltage adjustment © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Compensation theory Emission Optics © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. FITC Spillover PE 585/42 Relative Intensity FITC 530/30 500nm 550nm 600nm 650nm Wavelength (nm) © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 700nm FITC Compensation To Lower Cluster Increase % Subtracted PE 585/42 Relative Intensity FITC 530/30 20.10% of the Signal from FITC Sensed in PE 500nm 550nm 600nm 650nm Wavelength (nm) © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 700nm FITC Compensation © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Overcompensation vs Undercompensation Correct Compensation Overcompensation © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Undercompensation Compensation Examples Incorrect Compensation Correct Compensation Undercompensation © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Overcompensation Topic Introduction of Flow Cytometry Instrument setup Application of Flow Cytometry 44 © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Applications Phenotype Analysis (Cell Surface Antigens/Markers) Intracellular Analysis -- Eg. Cytokines, Signal Transduction molecules…etc. DNA Analysis -- Eg. Viability, Cell cycle, Apoptosis…etc. Cell Fuction Analysis -- Eg. Free radicals, Ca2+, Reporter genes…etc. CBA (Cytometric Bead Array) -- cytokine detection © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Phenotype Analysis Ligand Receptor Adhesion molecule …etc © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Lymphocyte Immunophenotyping Peripheral White Blood Cells CD45+ Monocytes Granulocytes Lymphocytes Monocytes Neutrophils B T CD3+ T Helper CD3+ CD4+ Confidential—For Internal Use Only T Cytotoxic CD3+ CD8+ CD3CD19+ HLA-DR+ NK CD3CD16+ CD56+ Basophils Eosinophils Lymphocyte Subset 48 © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Intracellular Analysis Cytokine Enzymes Structure Proteins Intracellular Signaling Confidential—For Internal Use Only Intracellular Analysis Permeabilizing solution Fixation solution © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Cell Surface and Cytoplasmic Stain protocol Confidential—For Internal Use Only Detection of Cell Surface and IC Cytokine CD4 FITC CD4 FITC © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. CD4 FITC Cell Function Analysis Membrane Potential (DiOC6, JC-1) Oxidative Metabolism (Free Radicals) Intracellular PH Value (Snarf-1) Ca++ Influx (Fluo-4/Fura Red, Indo-1) Phagocytosis Cell Proliferation (PI, BrdU, Intracellular Cyclins) Apoptosis (Annexin V, active Caspase-3) © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Cell Apoptosis 細胞凋亡 - Annexin V Apoptosis Assay PS © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Annexin V/PI Double Staining Early Apoptosis © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Cell Apoptosis 細胞凋亡 -Mitochondria potential change-JC-1 (BD Mitoscreen) Apoptosis通常與mitochondria的膜電位(Δψ)去極化有關性 JC-1 = J-aggregate-forming Cationic Δψ sensitive dye J-aggregates:膜電位Δψ 極化(polarized)時為此型式 JC-1 monomers: 膜電位Δψ (depolarized)去極化時存在此型式 J-aggregates 存於健康細胞,且螢光為FL1及FL2 channels (綠色及橘紅色螢光) JC-1 monomers通常但非絕對存於Δψ 去極化的apoptosis細胞,且只剩下FL1 (綠色螢光) © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Cell Apoptosis 細胞凋亡 - JC-1(BD Mitoscreen) Jurkat T cell data © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Cell Apoptosis 細胞凋亡 - MitoStatus TMRE/Red © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. DNA Analysis Ethanol Detergent Nucleic Acid Dye © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. DNA Dye Propidium NH2 7-AAD NH2 N+ C2H5 C2H2)3N+ CH3 C2H5 © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Cell Cycle Analysis G2 M G0 G0 G1 G1 S Count S 0 200 400 G2 M 600 4N 2N DNA content © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 800 1000 Apoptosis (Sub G1) © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Application of Flow Cytometry Soluble form protein detection Confidential—For Internal Use Only Cytometric Bead Array (CBA) © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Beads Provide a Flexible Platform Different fluorescence intensities Different colors with different intensities © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Cytometric Beads Array (CBA) © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 100 10 Simultaneous Analysis of Multiple Cytokines 1 10000 IL-2 IL-4 IL-5 IL-10 TNF-a IFN-g 1000 1000 100 100 10 10 1 1 10000 1000 1000 1000 100 100 100 10 10 0 pg 1 20 pg 1 10 40 pg 1000 100 100 10 1 312 pg 625 pg 10 10 100 1000 1 1250 pg 2500 pg 10 100 1000 5000 pg 1 11 1000 156 pg 1 1 1000 80 pg 10 10 100 100 1000 1000 11 10 10 10 100 100 100 1000 1000 10000 1000 10 100 1000 10000 FL2 100 10 1 10 trademarks 100 1000 1000 are property of Becton, Dickinson 11 all other 10 100 1 10 Company. 100 1000 10000 1 © 2016 BD. BD, the BD Logo and and 10 100 1000 FL3 Standard Curves IL-2 PE MFI:567 IL-2: 625pg/ml © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. BD Pharmingen Product list Cat# Name BD Pharmingen™ 550285 PI/RNase Staining Buffer 556463 BD Pharmingen™ Propidium Iodide Staining Solution BD Pharmingen™ 556419 Annexin V 556547 Size The reagent is suspended in a phosphate-buffered solution (pH 7.2) with 0.02% (w/v) sodium azide 100ml Propidium Iodide Staining Solution (For AnnexinV/PI assay use) 2ml Annexin V-FITC 200 test BD Pharmingen™ Annexin V Annexin V-FITC, Propidium : FITC Apoptosis Detection Iodide Staining Solution, Kit I Annexin V Binding Buffer 100 test BD Pharmingen™ 100 test 551302 BD™ MitoScreen (JC-1) 70 Content JC-1 dye and assay buffer BD Pharmingen™ BD Pharmingen™ MitoStatus 564696 MitoStatus TMRE TMRE BD Pharmingen™ MitoStatus 564697 BD Pharmingen™ © 2016 BD. BD, the BD Logo and all other trademarks Company. MitoStatus Redare property of Becton, Dickinson and Red 25mg 100ug Thank you! 71 張舒婷 新加坡商必帝公司 生物科學部 Product Specialist 產品專員 Mail: [email protected] Website: www.bdbiosciences.com/tw Confidential—For Internal Use Only