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1 BASIC PRINCIPAL AND APPLICATION OF CYTOFLEX / FC500 FLOW CYTOMETRY 美商貝克曼庫爾特生命科學產品專員陳盈諭 Michelle Chen [email protected]

2 Basic Principle Flow Cytometry is Made of 4 Systems 1. Fluid system 2. Optical system 3. Electronic system 4. Computer system Current Applications of Flow Cytometry 2

3 Study the Cells: Microscopy True Profile of the Cell Labor-intensive Arbitrary results 3

4 Why Use Flow Cytometry? Flow = Fluid Cyto = Cell Metry = Measurement 4

events to be acquired Simultaneous multicolor analysis (5

5 Characteristics of Flow Cytometry High speed acquire : ~3,300 ~30,000 events per second Up to 10,000,000 of total events to be acquired Simultaneous multicolor analysis (5 colors/10 colors) Higher accuracy, objective and more sensitive 5

6 Basic Principle Flow Cytometry is Made of 4 Systems 1. Fluid system 2. Optical system 3. Electronic system 4. Computer system Current Applications of Flow Cytometry 6

7 1. Fluid System: Hydrodynamic Focusing ( 流體動力聚焦 ) (ddh 2 O) (sample tube) ( ~10 7 / ml) Flow Cell 7

8 1. Fluid System: Hydrodynamic Focusing ( 流體動力聚焦 ) Sheath Max.~3,300 events / second Sample 8

9 CytoFLEX Sample Fluidics 吸樣前會自動攪拌可設定攪拌時間 Accepts 12x75mm and Eppendorf tubes Minimum Sample Volume: 10 µl 3 Pre-set Sample Rates Slow : 10 µl/min Medium : 30 µl/min Fast : 60 µl/min Adjustable Sample Rate: 10 µl µl/min Max.~30,000 events / second 9

10 Basic Principle Flow Cytometry is Made of 4 Systems 1. Fluid system 2. Optical system 3. Electronic system 4. Computer system Current Applications of Flow Cytometry 10

Principle of Flow Cytometry Sample Single cell suspension (2 10 5 ~10 7 / ml) Flow Cell ( 流動室 ) Sheath 405nm Violet Laser 488nm Blue Laser 638nm Red Laser Cross Section sample

11 Principle of Flow Cytometry Sample Single cell suspension ( ~10 7 / ml) Flow Cell ( 流動室 ) Sheath 405nm Violet Laser 488nm Blue Laser 638nm Red Laser Cross Section sample Light Scatter Forward Scatter (FS FSC) Side Scatter (SS SSC) Fluorescence (FL) FL1 FL4 (488nm laser) FL5 FL7 (638nm laser) FL8 FL10 (405nm laser) sheath Flow cell Orifice Waste 11

12 Forward Scatter (FSC) vs. Side Scatter (SSC) Larger side scatter LASER Larger forward scatter LASER Smaller side scatter Smaller forward scatter Forward scatter size of cells Side scatter granularity of cells 12

13 SSC granularity Example 3 P1 1 2 Grans Whole blood sample RBC lysed Monos Size: Lymphs Grans > Monos > Lymphs Granularity: FSC Size Grans > Monos > Lymphs 13

FC500 Dyes 使用 Channel FL1, 525+15nm FL2, 575+15nm FL3, 620+15nm FL4, 675+15nm FL5, >755nm 488nm Blue Laser FITC, Alexa Flour488 PE ECD, PE-Texas Red, PerCP

14 FC500 Dyes 使用 Channel FL1, nm FL2, nm FL3, nm FL4, nm FL5, >755nm 488nm Blue Laser FITC, Alexa Flour488 PE ECD, PE-Texas Red, PerCP PE-Cy5, PE-Cy5.5 PerCP-Cy5.5 PE-Cy7 GFP YFP PI (DNA) 7-AAD (DNA) 633 nm Red Laser APC APC-eFluor 647 Cy5 APC-Cy7 APC-eFluor

15 FC Optical System: Laser & Filter Set 488 nm Argon Laser 635 nm He-Ne Laser Side Scatter Forward Scatter Sample 15

16 FC Optical System: Filter Set 16

17 675 BP 620 BP FC Optical System : Filter Set Side Scatter 500 LP FL3 FL1 525 BP FL4 575 BP 755 BP BandPass (BP) Dichroic LongPass (DLP) Dichroic ShortPass (DSP) FL2 FL5 17

18 CytoFLEX Lasers & Dyes Laser 488nm Filter Channel Names Dyes 525/40 FITC FITC, AlexaFluor TM 488, CFSE, Fluo-3 585/42 PE PE, PI 690/50 PC5.5 PerCP, PerCP-Cy5.5, PE-Cy5, PE-Cy /60 PC7 PE-Cy7 660/20 APC APC, AlexaFluor TM 647, efluor TM nm 712/25 APC-A700 APC-A700, AlexaFluor TM /60 APC-A750 APC-A750, APC-Cy7, APC-H7, APC-eFluor TM /45 PB450 Pacific Blue, V450, efluor TM 450, BV421, DAPI, Hoechst 405nm 525/40 KO525 Krome Orange, AmCyan, V500, BV /20 Violet610 BV605, Qdot 605, ef605

19 CytoFLEX 2. Optical System: Laser & Filter Set Optical Fiber 488nm 638nm 405nm Side Scatter Flow Cell Forward Scatter Sample 13 19

20 CytoFLEX 2. Optical System: Filter 20

21 CytoFLEX 2. Optical System: Filter Set WDM Technology 多波長光分配模組 (Wavelength Division Multiplexing) Band pass filters APDs (Avalanche Photodiodes) Mirror Fiber optic cable 21

22 Basic Principle Flow Cytometry is Made of 4 Systems 1. Fluid system 2. Optical system 3. Electronic system 4. Computer system Current Applications of Flow Cytometry 22

23 3. Electronic System: 光電倍增管 (Photomultipliers, PMT) & Avalanche Photo Diode (APD), 放大訊號 (High Voltage) Detector 23

24 3. Electronic System: Discrimination or Threshold ( 排除雜訊 ) 垃圾桶垃圾桶 24

25 3. Electronic System: 將線性訊號轉換成對數訊號 FSC SSC Lin FL1 FL10 Log 25

26 線性訊號 (Lin) 對數訊號 (Log) Surface marker 例外 : Cell Cycle Platelets 26

27 Basic Principle Flow Cytometry is Made of 4 Systems 1. Fluid system 2. Optical system 3. Electronic system 4. Computer system Current Applications of Flow Cytometry 27

28 Flow Raw Data: FSC SSC 28

29 Data Displaying FSC SSC Dot Plot 29

30 Data Displaying FSC SSC Histogram Plot 30

31 Gating P2 P1 Histogram Plot Dot Plot 31

32 Computer System : 多色實驗多層次 Gating 32

33 DATA ANALYSIS :PLOT &STATISTICS 33

34 Plots 1. Single parameter histogram 34

35 1. Single parameter histogram overlap 35

36 2. Dual parameter histogram (1) Dot plot 36

37 2. Dual parameter histogram (2) Contour plot ( 等高線圖 ) 37

38 2. Dual parameter histogram (3) Density plot 38

39 Statistics 1. Percentage : % Total & %Gate 39

40 2. Mean : X-mean and Y-mean 40

41 3. Coefficient of Variant : CV ( 變異係數 ) 41

42 Flow Cytometer Experiment Settings 42

43 1. TWO COLOR EXPERIMENT SETTING Two color stain : FITC/PE Control Treat Flow Protocol Setting Sample Negative Control (Unstain) Positive FITC Positive PE Control (2-color) Treat (2-color) 43

44 Protocol Setting - Lymphocyte Immunophenotyping 上樣前 : 上檢體 : 1. Choose Parameter 2. Create Displaying Plot 3. Setting Discrimination/Threshold 4. 利用 Negative Control 調整 Voltage Gain 5. 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 44

45 CytoFLEX 1. Parameter Choice ( 選擇想要收取的參數 ) 45

46 CytoFLEX 2. Create Plot ( 利用已選定的參數來做圖 ) 46

47 CytoFLEX 3. Threshold( 設定雜訊排除的條件 ) 47

48 CytoFLEX 4. 利用 Negative Control 調整 Gain 48

5. 利用單染檢體調整 Compensation( 螢光補償 ) LASER 488

49 5. 利用單染檢體調整 Compensation( 螢光補償 ) LASER FL1 FL2 FL3 FL4 FL5 49

50 Compensation ( 螢光補償 ) FL1 PMT:92=90+2 FL2 PMT:100=

51 CytoFLEX 利用單染檢體調整螢光補償 1. 單染 FITC: 調整 FL2-%FL1 51

52 CytoFLEX 利用單染檢體調整螢光補償 2. 單染 PE: 調整 FL1-%FL2 52

53 CytoFLEX 利用調整好的條件正式上檢體 P1 53

54 CytoFLEX Compensation Library Automatic and manual Store lot-specific dye information Auto adjustment Absolute Linearity in gain adjustment

55 Basic Principle Flow Cytometry is Made of 4 Systems 1. Fluid system 2. Optical system 3. Electronic system 4. Computer system Current Applications of Flow Cytometry 55

56 Cell Surface Marker Immunofluoresecnce analysis Leukocyte Immunophenotyping ( 淋巴細胞免疫分型 ) Stem cells tracking and enumeration ( 幹細胞分析及計量 ) Analysis of platelets ( 血小板功能分析 ) Cell function assays Cell Cycle and Apoptosis Mitochondrial membrane potential analysis (DiOC6 JC-1) Calcium kinetic studies (Fluo-3) Cellular protein content measurements (GFP YFP) NK cell cytotoxicity assay (CFSE+ PI ) Phagocytosis assay (FITC-E. coli BioParticles + PI) Extracellular cytokines detection (beads array) Microorganisms analysis 56

57 Lymphocyte Markers Antigen CD3 CD3+CD4 CD3+CD8 CD16+CD56 CD19 or CD20 CD25 CD38 CD45 CD45RO+CD4 or CD8 CD45RA+CD62L+CD4 or CD8 Target cell recognized Pan T cell T helper cells T cytotoxic cells NK cells B cells Activated cells Activated cells Pan leukocyte Memory T cells Naïve T cells 57

58 Lymphocyte Immunophenotyping T cell / B cell Helper T cell NK cell / Suppressor T Cytotoxic T cell 58

59 9 color T Cell Receptor Characterization of PBMCs 59

60 Rare Events-Low Expressed Regulatory T cells Human PBMC- regulatory T cells 60

61 Hematopoietic /Progenitor Stem cell (HPC) identification and enumeration( 幹細胞計數 ) CD45-FITC CD34-PE 7-AAD 61

HO33342 blue fluorescence side scatter side scatter APC-Cy7 c-kit Side Population

marrow Hoechst 33342 at 5 μg/ml (blue = 450/40 nm, red = 675/20 nm) NUVLD 375 nm

APC Lineage (660/20 nm) forward scatter APC lineage PE-Cy7 Sca-1 A B C Lineage 1.

2% Sca-1+ c-kit+ HO33342 red fluorescence Identification of side population from

62 HO33342 blue fluorescence side scatter side scatter APC-Cy7 c-kit Side Population DC1275 Hoechst SP on BC CytoFLEX S 3 color sample 129/C57B/6 mouse bone marrow Hoechst at 5 μg/ml (blue = 450/40 nm, red = 675/20 nm) NUVLD 375 nm excitation PE-Cy7 anti-mouse Sca-1 (780/60 nm) APC-Cy7 anti-mouse c-kit (780/60 nm) APC Lineage (660/20 nm) forward scatter APC lineage PE-Cy7 Sca-1 A B C Lineage 1.45% all 2.68% negative 57.2% Sca-1+ c-kit+ HO33342 red fluorescence Identification of side population from murine bone marrow: utilizing Dye Cycle Violet exclusion (A, all) are lineage negative (B) and lineage negative AND double positive for sca-1 and c-kit (C)

63 Analysis of Platelets Platelet activation marker P-selectin (CD62P). 63

64 Mitochondrial membrane potential analysis (DiOC6) 64

65 Cell cycle and DNA content analysis Propidium Iodide ( PI ) A fluorescent dye is bound directly to the DNA in the nucleus of cells. Measuring the fluorescence provides a measure of the amount of dye taken up by the cell and indirectly the amount of DNA content. Other DNA Dye : 7-AAD PI emits at 610 nm ( FL 3 ) Acridine Orange (AO) YOYO-1 65

66 Cell Cycle Analysis with 488nm Laser Drug Effect on Cell Cycle 2C 4C Control Colchicine Camptothecin Anti-CD95 秋水仙素抗癌藥物 - 抑制 DNA 複製誘導 apoptosis 66

檢測細胞凋亡早期反應 : Annexin V 檢測細胞凋亡細胞凋亡早期改變發生在細胞膜表面, 這些細胞膜表面的改變之一是 Phosphotidyl Serine(PS) 磷脂從細胞膜內轉移到細胞膜外, 使 PS 暴露在細胞膜外表面在細胞發生凋亡時, 細胞膜上的這種磷脂分佈的不對稱性被破壞, 而使 PS 暴露在細胞膜外 Annexin V 是一種 Ca ++ 依賴的磷脂結合蛋白,

67 檢測細胞凋亡早期反應 : Annexin V 檢測細胞凋亡細胞凋亡早期改變發生在細胞膜表面, 這些細胞膜表面的改變之一是 Phosphotidyl Serine(PS) 磷脂從細胞膜內轉移到細胞膜外, 使 PS 暴露在細胞膜外表面在細胞發生凋亡時, 細胞膜上的這種磷脂分佈的不對稱性被破壞, 而使 PS 暴露在細胞膜外 Annexin V 是一種 Ca ++ 依賴的磷脂結合蛋白, 具有易與磷脂類 ( 如 PS) 結合的特性, 對 PS 有高度的親和性, 因此該蛋白可作為一敏感的探針來檢測暴露在細胞膜表面的 PS 然而 PS 轉移到細胞膜外不是凋亡所獨特的, 也可發生在壞死細胞上兩種細胞死亡方式間的差別是在凋亡的初始階段細胞膜是完好的, 而細胞壞死在其早期階段細胞膜的完整性就遭到破壞因此 Annexin V 檢測法多搭配一種 PI 或 7-AAD 染料排除試驗來達到同時辨識壞死細胞與凋亡細胞的實驗目的 67

68 檢測細胞凋亡早期反應 : Annexin V 檢測細胞凋亡 Apoptosis Annexin V+ PI+ (late apoptotic/necrotic) Annexin V+ PI- (early apoptotic) Jurkat Cells, induced with Camptothecin overnight at 37ºC (treated) 68

69 檢測細胞凋亡晚期反應細胞凋亡晚期中, 核酸內切酶在核小體之間剪切核 DNA, 産生大量長度在 bp 的 DNA 片段對於這一現象的檢測通常有以下兩種流式檢測方法 : 1. Sub-G0/G1 phase ( 亞二倍體期 ) Control Drug treated 69

70 E.coli 周期倍體數檢測 70

71 Nano Particles Vesicles Prepared from Rat Plasma PBS ONLY Vesicles AnnV Dye 488 CD42d Dye 488 Vesicles prepared from Rat plasma. Treated versus untreated, using both AnnexinV and CD42d Dy488. Data provided by John Nolan, Scintillon Inst.

72 Beadless volumetric counting for single platform enumeration Absolute Cell Counts 72

73 QUESTIONS 73

74 THANKS FOR YOUR ATTENTION! 74

neutral red photodetector Andrew Moldavan 1960 Louis Kamentsky Moldavan microscope-based spectrophotometer 500 ultraviolet absorption scatter of blue

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