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1 Principle of Flow Cytometry and Outline of EC800 Cell Analyzer

2 What is Flow Cytometry Flow Cyto Metry Cells in Motion Cell Measure You can get the characteristics of cells by measurement various light in the flow

3 Flow Cytometer Grouping of Flow Cytometry Products Cell Analyzer Data acquisition and analysis Auto sample loader Cell analyzer SP6800Z Cell Sorter Data acquisition and analysis Cell sorting (collection) Cell analyzer EC800 Cell Sorter SH800Z

4 Basics of Flow Cytometry 4 6/9/2015 Fluidics: Optics Electronics Computing cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered and converted to digital values that are stored on a computer Original Slide from Bob Murphy, CMU

5 Principle of Flow Cytometry Fluidics Optics Detectors Electronics Computing Bandpass Filters Dichroic Mirrors If laminar flow made in flow cell, cells are made to pass the center of sheath stream forcibly by using the hydrodynamic focusing Cells are detected one by one with laser beam Various emitted light are efficiently collected, separated by optical filters and detected with PMTs Detectors output voltage signals related to brightness 5 6/9/2015 Signals are converted to digital value

6 Conceptual scheme of Flow Cytometer Fluidics Electronics Flow Cell Optics and Detectors Computing From :

7 Fluidics Flow Cell Fluidics System Flow Cell Sheath Sample Sheath Difference of the pressure Sample and Sheath makes Hydrodynamic Focusing in the Flow cell. Sample flow is very narrow, so cells line up and flow at constant velocity. Laser Cell

8 Optical System Optics and Detectors Separate fluorescence based on wavelength Flow Cell Dichroic long pass filters (DLP) Detectors for Fluorescence (PMT) When a cell passes a laser beam in flow cells, the cell emit scatter and fluorescence from the fluorescent dye. Forward Scatter (FSC), Side Scatter (SSC) and fluorescence (FL) are separated based on their wavelength by optics filters and lead to detectors (PMT). Band pass filter (BP) Pass only limited wavelength light FSC Red Yellow Green SSC From:

9 Separation of Fluorescence by Optical Filters For Example : Red fluorescence(660nm) a c a. 639nm DLP pass it ( 660nm > 639nm) b. 752nm DLP reflect it ( 660nm < 752nm) b d c. 685nm DLP reflect it ( 660nm < 685nm) d. 665/30nm BP(665±15nm) pass ( 650nm <660nm < 680nm) e e. FL4(PMT) detect it FL4 PMT

10 Multi Laser / Multi Color 405nm 561nm 488nm 638nm Multi Laser gives you flexibility choice of Flourochromes.

11 Multi-Laser / Multi-Color Can provide you various information of cells at once Laser Flourochromes Max. Emission Band pass filter

12 Electronics / ADC / Plot Voltage pulse Height Area Time Histogram The scatter and the fluorescence occur when a cell passes a laser beam. Detectors make out voltage pulse caused from each intensity. Pulse Height are directly proportional to intensity. However, they change by detector's sensitivity control. Integrated value (Area) shows total amount of light from the cell. These analog value are converted to digital value by ADC. Cytogram

13 Visualization of the cell poplations Make plots, identify the cell populations Number of cells Parameter (Log axis) Parameter (Log axis) Parameter (Log axis) Number of cells Parameter (Lin axis) Histogram X axis : Intensity of light Parameter (Lin axis) Parameter (Lin axis) Cytogram Show correlation of two parameters (light). Dot density = population density.

14 フローサイトメーターで測定しよう

15 Samples available on FCM Cells (micro particle suspension) Size : 0.5 μm ~40 μm Concentration : 1x10 6 ~5x10 6 個 /ml Blood cells Cell line Pant Bacteria Marker (cell surface, intra culler) DNA content (Cell cycle, Diploidy) Cell kinetics (Active oxygen, etc.) Cell death (Apoptosis) Gene expression (Fluorescence protein) Marline life Cytokine etc. Intra culler stain, Capture beads (Cytometric Bead Array : CBA)

16 Limit of the form identification WBC Monocytes Granulocytes Lymphocytes What are the markers for lymphocyte subsets?

17 Parameters on FCM For Individual cell 1. Scatter Forward Scatter (FSC) Cell size Side Scatter (SSC) Internal structure of the cell 2. Fluorescence Dyes 3. Time 4. others (Ratio, EV, etc.)

18 Laser Scatter Forward Scatter : FSC = relevant to cell size * Side Scatter : SSC = relevant to Internal structure of the cell

19 Scatter plot (FSC vs SSC) WBC sample Complex SSC Lin 顆粒球 リンパ球 単球 Internal structure FSC Lin Simple small Size Large

20 More ~Fluorescence cell surface marker There are many protein molecules on cell surface Dye conjugated Monoclonal Antibody bind to them specifically. membrane

21 Immunophenotyping More ~Fluorescence Cell surface marker (antigen) Dye conjugated antibody Emit from dye CD No. Excitation Emission

22 Dye conjugated antibody Emit fluorescence = AB bind to cell = Positive Negative Dim Positive Bright Many antibody bind to the cell that express many antigen.

23 FL Histogram Negative Posotive Dim Bright Cell numbers dim intensity bright

24 2 FL Cytogram PE Double positive Single positive (PE) /+ +/+ Double negative +/ / FITC Single positive (FITC) P. 24

25 Many fluorescence dyes M G2 G1 G0 S GFP There are many many fluorescence dyes : For DNA : Cell Cycle analysis, Apoptosis, etc. (DAPI, PI, etc.) Activated Oxygen, ph, Calcium con. Etc. : Kinetics analysis (Flou-3, Flou-4, etc.) Gene expression : Fluorescence protein : GFP,CFP, YFP, mcherry, etc.

26 FCM applications Kinetics analysis DNA/RNA analysis Immunophenotyping フローサイトメトリー Death, Apoptosis sorting

27 Immunophenotyping~Lymphocytes subset analysis リンパ球は以前に増して機能的な多数のサブセットに分 類されるが形態学的にこれらのサブセットを識別する ことは困難であり フローサイトメトリーにより機能的な 各サブセットの目印 ( マーカー ) となるものを指標として, サブセットの分類や解析を行う Lymphocytes Monocytes T Cell Cell type B Cell NK Cell Monocytes T helper T Cytoxic CD2, CD3, CD2, CD3, CD5, CD2, CD14 CD5, CD7, CD5, CD7, CD19, CD7, Typical 代表的マーカー CD4 CD8 CD20, CD16, marker CD21 CD56 CD:Cluster of differentiation

28 Immunophenotyping~Lymphocytes subset analysis Multi-Color analysis : setting of positive region Positive region made refer to Negative control 陰性コントロール IgG のみ % of positive region

29 Immunophenotyping~Lymphocytes subset analysis Gating G FS L M Make a gate region around lymphocytes population Lymphocytes 全体

30 Immunophenotyping~Lymphocytes subset analysis (No gate = all events) (Only in the gate) (incorrect gate) 30

31 Immunophenotyping~Lymphocytes subset analysis Multi-Color analysis T 細胞 B 細胞 NK 細胞 CD2 CD3 CD2 CD3 CD3 CD2 CD19 CD16 CD5 CD5 CD5 CD7 CD7 Th CD4 CD4 Tc CD8 CD8 CD7 CD20 CD56 FITC PE PE Cy5 Lym subset CD3 FITC CD56 PE Cy5 T 細胞 subset CD8 FITC CD3 PE Cy5

32 Immunophenotyping~Lymphocytes subset analysis Multi-Color analysis : Compensation Fluorescence emission is very broad. The part of it overlap on other detectors. It is necessary to set the compensation for view the data plot correctly. Pacific BV421 Blue FITC PE APC PerCP/ Cy5.5 PE/Cy7

33 FITC overlap to PE detector, PE overlap to FITC detector. PE s emission No compensation Detection by FL2 (PE) FL2 FL1 FL2 Band pass Detection by FL1 (FITC) FITC s emission FL1

34 FITC overlap to PE detector, PE overlap to FITC detector. PE s emission Compensation are applied. subtraction FL1 FL2 Band pass filter subtraction FITC s emission Compensation Matrix

35 Immunophenotyping~Lymphocytes subset analysis FITC Single positive control sample Multi-Color analysis : Compensation Under compensation Good compensation Over compensation

36 Immunophenotyping~Lymphocytes subset analysis Multi-Color analysis : Select of Fluorescence Dye Point Negative Performance definition CD4/FITC 1. Reactivity 2. Antibody reagent Antibody clone, maker, product lot. Vendor Positive Median values Resolution Positive Negative M(pos) / M(neg) 3. Conjugated Dye 4. Check the overlaps. A 217,4 3,4 64 B 105,6 3,18 33 C 162,8 3,29 50

37 Immunophenotyping~Lymphocytes subset analysis Multi-Color analysis : Resolution CD3-FITC CD3-PE CD3-APC Emission intensity are different between each dyes. Stain Index = D/W D = 陰性ピークと陽性ピークの中央値 (Median) の差 W = 2 x rsd (robust standard deviation) Holden Maecker & Joe Trotter, Nature Methods 5, (2008)

38 Immunophenotyping~:T Reg identify 38 6/9/2015 T reg phenotype CD3 FITC CD4 APC CD25 PE Cy7 CD127 PE

39 DNA analysis~cell cycle Classic and important application Cell Cycle=increase of the cell M G0~G1~S~G2~M~G1~S~G2~M~G1~ G2 G1 G0 S Stain with DNA binding dye (PI, etc.) Fluorescence Intensity DNA Content (intensity of G2 phase cells are 2 times G0/G1 phase cells) Tumor cells often aneuploidy.

40 DNA analysis ~ Cell cycle analysis Technical Tip1: Doublet discrimination Height Width All events Heig ht Area Only in Gate

41 DNA analysis ~ Cell cycle Technical Tip 1: Doublet discrimiation 蛍光パラメーターのパルスAreaおよびwidthを用いる電気的ダブレット除去により 2つの細胞が結合しているダブレットや細胞凝集物によるデータのアーティファクトを除くことができる 1. 細胞間のわずかな差 (DNA 等 ) を定量的測定の精度 2. 解析データの直線性および正確さ Area 3. ソーティング時の純度 の向上につながります Width From : protocol.org/e973

42 Laser Sample pressure : High Laser Sample pressure : Low sample/sheath DNA analysis ~ Cell Cycle analysis Technical Tip 2: sample pressure v.s. CV event event CV(%)= 100 (Peak の標準偏差 )/(Peak の平均値 ) FL Lin FL Lin るアプリケーション High Flow Rate > CV large 適用 : 細胞マーカー解析 ( 免疫蛍光法 蛍光抗体法 ) 蛍光 タンパク質発現解析など 蛍光データを Log 表示で解析す Low Flow Rate > CV small 適用 : Cell Cycle Analysis( 細胞周期解析 ) など 蛍光データ を Lin 表示で解析するアプリケーション

43 Gene expression ~ Fluorescence protein 目的の遺伝子に蛍光タンパク遺伝子を連結し 細胞に導入 目的遺伝子が発現すると 蛍光タンパク遺伝子も発現 蛍光タンパクがある細胞 = 遺伝子発現細胞 遺伝子発現細胞の蛍光標識 目的遺伝子を発現する細胞を解析 ソーティング GFP DsRed HcRed CFP CFP DsRed レーザー :405nm, 488nm, 561nm

44 Micro biology ~ cell count bacteria/yeast E. coli BSC-A BSC-A FSC-A FSC-A 例 : 細菌計数アッセイ : 細菌の同定に使用する複数の蛍光核酸染色剤の混合液と正確なサンプル量の測定を可能にする校正済みビーズ懸濁液が含まれている 44 6/9/2015

45 Intra celluler protein ~Cytokine assay Intra celluler Cytokine Staining Assay 特長 : 刺激による細胞のサイトカイン産生能力を検出できる 細胞表面マーカーと組み合わせることで ヘテロなサンプル中の特定のポピュレーションを絞って観察することも 異なるポピュレーションを比較することも可能 同時に複数のサイトカインを検出することができる 45 6/9/2015

46 フローサイトメトリーによる実験の流れ 準備段階 使用する抗体 蛍光色素の割り当てを計画 細胞の準備 校正用サンプルの準備 フローメーターの最適化 シース流の安定化 ソーティングの場合は液滴形成 偏向電圧の調整 PMT 検出器電圧の最適化 蛍光漏れ込み補正 標的目標の解析 分取用ゲートを決定 サンプルの解析 ソーティング ソーティング時の細胞濃度に注意 液滴の安定性をモニタリング 実験目的にあわせて回収容器を選別

47 フローサイトメトリーの特徴 高速測定 : 最大数万細胞 / 秒での客観的測定が可能 マルチパラメータ解析 : 複数蛍光色素 ( ラベル ) の組合せにより多重パラメータデータを取得 & 解析できる 高再現性 : 同一条件での測定が可能 高速分取 : 測定結果に従い 特定の細胞集団を生きたまま分取可能

48 SONY FCM Products

49 From Blu-ray to Flow Cytometer 49 6/9/2015

50 From Blu-ray to Flow Cytometer Blu-ray Hi-Speed spin Flow Cytometer Hi-Speed flow Cell Lens Laser Lens Laser Zoom up of Disc 50 6/9/2015

51 Next Generation Cell Sorter Easy to use for everyone Conventional cell sorters require adjustment of optical axis, droplet formation, side stream adjustment, decision of delay time by a dedicated operator. However, SH800Z eliminates the need for these tasks. Also, the adoption of a plastic chip sorting, washing operation is no longer time-consuming. In addition, we have achieved a small 1/3 compared to conventional products by taking advantage of the integrated technology and compact mechanical design technology cultivated in the Blu-ray optical techniques. As "personal" cell sorter, the SH800Z will contribute to improvement of the workflow efficiency, and cost reduction for everyone including customers who heavily use the sorter for their research. Cell Sorter SH800Z

52 Advantage 1.Full automatic setup about 10 min. no user s adjustment unnecessary the FCM specialist 2.Easy maintenance disposable sort chip quick change of sample line no carriover 3.Compact body and 4 lasers W 55cm, D 55cm (1/3 of conventional sorter) 488,405,561,638nm P. 52

53 convenient affordable flow cytometry analysis One to four selectable and upgradable laser excitation options 405 nm, 488 nm, 561 nm, 642 nm Four to six PMT s, field upgradeable, with user changeable filters Any Fluorochrome Anywhere Flexible autoloader for 12x75 mm, PCR, or Eppendorf tubes, and more than 10 plate formats Flexibility at your fingertips, no extra charge Cell Analyzer EC800 53

54 1.Hybrid Flow cell (Laser and Electronics) EV Laser : Scatter(FS, SS), Fluorescence (FL1-FL5) EV (Electronic Volume) : Impedance FL1-FL5 FS SS 54 P. 54

55 EV v. s. FS 55 P. 55

56 Separation : Cells Debris (EV v. s. FS) Adhesion cell line sample data EV is better than FS 56 P. 56

57 2.Adjustment of gains is available after acquisition 24bit Wide dynamic ADC Touch screen 1000 times than conventional Digital gain adjustment Drag and Drop Drag and Drop 57 P. 57

58 Compensation : No affected by Digital gain adjustment Conventional Compensation PMT s Voltage Adjustment 58 P. 58

59 3.Full automatic sampler Syringe sampling system Auto probe cleaning Tube and Well plate are available Side holder for Reagents Pipetting for sample re-susupension 59 P. 59

60 Tube and well plate Tube : 1.5mL, 5mL Well plate (24, 96, 384 ウェル ) are available 60 P. 60

61 世界初 スペクトル型 フローサイトメーター The SP6800 Spectral Analyzer is Sony's newest innovative life science system fundamentally expanding the way cell and biomarker analysis can be performed. This system incorporates a unique optical bench, Blu-ray disc technology, and advanced algorithms to deliver some of the most accurate and precise data available. To improve accuracy of data, this system also provides unique functions to display and analyze cellular auto fluorescence and allows the user to easily automatically remove. 61 6/9/2015 Spectral Cell Analyzer SP6800

62 Revolutionary Optics System: SP6800 Unique Flow cell Chip 4ch PDA detects each event s location c High transparency Prisms 32ch array PMT Auto Alignment Prism Array Unique 32 micro lens array to improving optical efficiency Micro Lens Array 62 6/9/ Sony Biotechnology Inc. Engineering For The Future

63 Spectrum Plot FL Spectrum Sample flow Laser FL signal Intansity 個々の細胞の蛍光スペクトル SP6800 measure FL intensity with 32 PMTs 蛍光波 32ch PMT Spectrum Plot Intensity High Frequency. Wavelength Wavelength Overlay Spectrum curves of many cells Intensity 450nm 800nm 63 6/9/2015 Low Frequency. Wavelength (nm)

64 Sample Spectra Spectral Un-mixing Flow Cytometry Data SP6800 Algorithm c Reference Spectra Unstained AF 488 AF 532 PE PE-TR PE-Cy5 PerCP-Cy5.5 PE-Cy7 BV510 BV570 BV711 Average! Just like compensation /9/ Sony Biotechnology Inc. Engineering For The Future

65 SP6800 Advantages of Spectral Flow Cytometry 1. Adjacent Fluorescence are available at the same time. Super-Multi Color analysis Adjacent Fluorescence separation 2. Auto Fluorescence spectrum can be get! Auto Fluorescence subtraction Spectral Signatures (label free flow) c Control Auto Fluorescence Suimulated 3. It can support new debut Dyes flexibly. 65 6/9/ Sony Biotechnology Inc. Engineering For The Future

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