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1 Flow Cytometry & Cell Sorting 郑晓东

2 Content Basic of flow cytometry Application of flow cytometry

3 Basic of flow cytometry History Flow cytometry instrumentation Fluorochrome How do Flow cytometry work?

4 Basic of flow cytometry History Flow cytometry instrumentation Fluorochrome How do Flow cytometry work?

5 History Automatic fluorescence antibody Cell Counter Flow cytometry

6 BD FACSCalibur

7 BD FACSAria

8 BD LSRII

9 Basic of flow cytometry History Flow cytometry instrumentation Fluorochrome How do Flow cytometry work?

10 Flow Cytometry Instrumentation Fluidic systems Light Sources & Optical systems Electronic Measurements & Signal Processing Sorting Data collection and analysis

11 Flow Cytometry Instrumentation Fluidic systems Light Sources & Optical systems Electronic Measurements & Signal Processing Sorting Data collection and analysis

12 Sample and sheath fluid

13 Flow Cell Injector Tip Sheath fluid Fluorescence signals Focused laser beam

15 Flow Cytometry Instrumentation Fluidic systems Light Sources & Optical systems Electronic Measurements & Signal Processing Sorting Data collection and analysis

16 Light Sources - Laser

17 Light Sources - Laser

18 Light Sources - Laser FACSAria

19 Signal Collection

20 Long Pass Filters Transmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths greater than 500nm Transmittance 400nm 500nm 600nm 700nm

21 Short Pass Filter Transmits all wavelengths less than specified wavelength Example: 600SP will transmit all wavelengths less than 600nm. Transmittance 400nm 500nm 600nm 700nm

22 Band Pass Filter Transmits a specific band of wavelengths Example: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20) Transmittance 400nm 500nm 600nm 700nm

23 Dichroic Filter/Mirror Light Source Filter placed at 45 o Transmitted Light Reflected light

24 FACSCalibur optical layout

25 Flow Cytometry Instrumentation Fluidic systems Light Sources & Optical systems Electronic Measurements & Signal Processing Sorting Data collection and analysis

26 FACSCalibur optical systems

27 FACSAria optical systems

28 FACSAria optical systems

29 Signal Processing 1. Laser t Voltage 2. Laser t Voltage 3. Laser t Voltage

30 Voltage Pulse Height (H) Pulse area(a) 0 Pulse Width (W) 40 Time (µs) FLx-H: 萤光之电位脉冲高, 与细胞散发之平均萤光强度成正比, 一般用于表示表面抗原或是蛋白分子表达 ; FLx-A: 细胞散发之总萤光强, 以图形之积分面积表示, 多用於细胞周期 DNA 分析 ; FLx-W: 细胞通过射区所需之时间, 则以宽表示,, 可用区分单一个细胞与双细胞或多细胞体, 因为较大物件通过射区的时间较单个细胞所需之时间得长的原故.

31 Threshold The threshold defines the minimal signal intensity which has to be surpassed on a certain channel. All signals with a lower intensity are not displayed and not recorded for later analysis.

32 Flow Cytometry Instrumentation Fluidic systems Light Sources & Optical systems Electronic Measurements & Signal Processing Sorting Data collection and analysis

33 Fluorescence Activated Cell Sorting 488 nm laser FALS Sensor Fluorescence detector Charged Plates - + Single cells sorted into test tubes

34 Cell Sorting FACSAria

35 Flow Cytometry Instrumentation Fluidic systems Light Sources & Optical systems Electronic Measurements & Signal Processing Sorting Data collection and analysis

36 Data Acquisition - Listmode Event Param 1 Param 2 Param 3 Param 4 FSC SSC FITC PE

37 Data analysis software CellQuest (Mac):FACSCalibur WinMDI (PC):Free ModFit (PC, MAC):Cell cycle Diva (PC): FACSAria, FACSLSR FlowJo (PC, MAC):

38 Histogram Event # Param 1 FSC Param 2 SSC Param 3 FITC Param 4 PE Param 5 APC

39 Plots Contour Plot Density Plot Greyscale Density Dot Plot

40 Basics of Flow Cytometry Fluidics Optics Electronics 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

41 Fluorochrome Organic Fluorochrome: FITC, PE, GFP,CFP, YFP PE-CY5, PerCP-Cy5.5, APC-CY7, PE-CY7 Quantum Dots

42 Excitation and emission of fluorochrome Excitation Emission Laser

43 Excitation and emission of fluorochrome fluorescence resonance energy transfer (FRET)

44 Excitation and emission of fluorochrome

45 Stain Index = D/W

46 Choice of Fluorochromes PE- and APC-labeled antibodies be used for staining antigens that are expressed at relatively low levels. Fluorescein isothiocyanate (FITC)- and PerCP-labeled reagents should be used for staining antigens that are coexpressed at relatively higher levels. Flow cytometry: laser (488 nm, 643 nm), PMT

47 Typical instrument configuration

48 Quantum Dots

49 Optical Properties of QDs 发射波长与直径正相关, 可被通一激光激发 ( 通常 <450nm)

50 Optical Properties of QDs 激发波长与发射波长间距宽

51 Optical Properties of QDs 亮度高

52 Antifade

53 Basic of flow cytometry History Flow cytometry instrumentation Fluorochrome How do Flow cytometry work?

54 What Can a Flow Cytometer Tell Us About a Cell? Its relative size (Forward Scatter --FSC) Its relative granularity or internal complexity (Side Scatter--SSC) Its relative fluorescence intensity (FL1, FL2, FL3, FL4, and FL5, etc)

55 Forward Angle Light Scatter Laser FALS Sensor

56 Side Light Scatter Laser FALS Sensor Sensor

57 Light Scatter FSC( 前散射光 ) 它反应细胞的相对大小 SSC ( 侧散射光 ) 代表细胞的颗粒度的多少 Neutrophils Monocytes Lymphocytes

58 Fluorescence Detectors Laser FALS Sensor Freq Fluorescence Fluorescence detector (PMT3, PMT4 etc.)

59 Fluorescence FITC FITC FITC FITC FITC FITC FITC FITC

60 Important Points on Analysis Sample Gating Compensation Staining Controls

61 样品要求单细胞 / 颗粒悬液 : 细胞粘连可以导致检测误差被检细胞或颗粒大小 : um (FACScalibur) 由机器灵敏度和上样针的直径决定样品中至少个细胞, 最适浓度个 / 毫升

62 Gating No Gate Gated

63 Neutrophils Monocytes Lymphocytes CD4 CD3

64 Compensation

65 Compensation

66 Staining Controls Autofluorescence controls Isotype control Stain with an immunoglobulin (Ig) isotype control of irrelevant specificity. Compensation controls Positive Staining Controls

67 1. Autofluorescence controls Thymocytes Splenocytes 所有的细胞都有全波长的自发荧光在体外培养细胞肿瘤细胞或含颗粒较多的细胞与其他细胞比较可能有相对高的自发荧光为了检测每一种研究的细胞群的基本荧光, 未染色的细胞用于对照, 以排除自发荧光的影响 - 自发荧光对照目的是设定仪器 PMT 的检测电压, 让未染色的细胞处于左下角

68 Ig isotype control IgG2a-PE-CY5 CD8 IgG2b FITC CD4 Ig 同型对照 : 用与检测特异性无关的同种同亚型同荧光抗体染色作为同型对照 (Ig isotype control) 同型对照用于检测细胞与抗体非特异结合所至非特异染色

69 Compensation controls CD8 CD8-PE-Cy5 CD4-FITC CD4 当多种荧光被单波长激发时, 荧光发射光谱会产生重叠, 为纠正荧光光谱重叠 -Compensation controls 细胞样本分别用单独的荧光抗体染色 (Compensation controls), 来决定荧光重叠的水平, 确定适合的 Compensation

70 Content Basic of flow cytometry Application of flow cytometry

71 Application of flow cytometry Measurement of membrane Ag Measurement of intracellular Ag Cytometric Bead Array (CBA) Analysis of proliferation Analysis of cell apoptosis Sorting

72 Application of flow cytometry Measurement of membrane Ag Measurement of intracellular Ag Cytometric Bead Array (CBA) Analysis of proliferation Analysis of cell apoptosis Sorting

73 Detection of membrane Ag CD molecules Receptor: Cytokine Receptor, Protein Tyrosine Kinase Receptor, TNF Receptor, Toll-like Receptor, Chemokine receptors Antigen-specific marker: Tetramer Function marker: Annexin V

74 Protocol: Multicolor Immunofluorescent Staining for Cell Surface Antigens. 1. Harvest cells Preparing suspensions of single cells: Blood cells, Cells from tissue, Culture cells 2. Block Immunoglobulin Fc Receptors. Reagents that block immunoglobulin Fc receptors (FcR) may be useful for reducing nonspecific immunofluorescent staining. The cells are not washed before the first staining step. 3. Stain for Cell Surface Antigens Direct immunofluorescent staining: Fluorochrome-antibody Indirect immunofluorescent staining: 2nd antibody, streptavidin-biotin 4. Acquired and Analysis by FACS

75 Thymocytes double staining PBS DH5 CD8 CD4 CD4

76 Flow cytometric analysis of antigen-specific T cells

77 Application of flow cytometry Measurement of membrane Ag Measurement of intracellular Ag Cytometric Bead Array (CBA) Analysis of proliferation Analysis of cell apoptosis Sorting

78 Detection of intracellular Ag Preparing suspensions of single cells Staining the membrane protein Fixation Permeabilization Blocking Staining the intracellular Ag

79 Intracellular Cytokines IFN- IL-4 CD3 CD4

80 Phosphorylation Signaling Protein

82 Cytometric Bead Array (CBA) Capture beads Fluorescence Detector antibody Standard Protein

83 How to analyze soluble proteins in FCM? Beads Capture antibody Capture beads + + Antigen (cytokines) Fluorescence Detector antibody

84 Multiplexed Beads Various analytes Antibody coupled beads, emitting at distinct FL3 intensities Antibody coupled PE label, emitting at FL2 intensity proportional to analyte conc. Shades of a color

85 Beads Provide a Flexible Platform Beads provide an expandable assay platform for use with a flow cytometer Different intensities Multiple sizes Different colors with different intensities

86 Bead Array

87 CBA Standard curves

88 Analysis of proliferation PI BrdU CFSE

89 Cell Cycle G 2 s M G 0 G 1 DNA Analysis G 0 G 1 Count s G 2 M N 4N DNA content

90 Detection of Cell cycle (PI staining) Preparing suspensions of single cells Resuspend cell pellet in 300ul of cold PBS. Fix cells by adding 700 ul of -20 o C absolute ethanol. (drop by drop initially) Store cells at -20 C in this fixation buffer until ready for analysis. (less than one month) Centrifuge fixed cells and resuspend pellet in 500 ul of PBS. Add 100 ul of 200 ug/ml DNase-free, RNaseA and incubate at 37 o C for 30 min. Add ul of 1 mg/ml PI and incubate at room temperature for 5-10 min. Analyzed by FACS.

91 A typical DNA Histogram of cell proliferation

93 BrdU labeling Events Brdu,5- 溴脱氧尿嘧啶核苷, 为胸腺嘧啶的衍生物, 可代替胸腺嘧啶在 DNA 合成期 (S 期 ), 活体注射或细胞培养加入, 而后利用抗 Brdu 单克隆抗体, 显示增殖细胞同时结合其它细胞标记物, 双重染色, 可判断增殖细胞的种类, 增殖速度, 对研究细胞动力学有重要意义

94 CFSE

95 Analysis of apoptosis PI Annexin V

96 Flow Cytometry of Apoptotic Cells Apoptotic cells Normal G0/G1 cells Events PI - Fluorescence

97 Classical Technique: Annexin V Assay Annexin V-FITC conjugate C a++ C a++ C a++ C a++ Apoptosis Plasma membrane Externalization of phosphatidylserine Cytoplasm Cytoplasm

98 Detection of Cell apoptosis Induce apoptosis by desired method. Collect 1-5 x 10 5 cells by centrifugation. Resuspend cells in ul of Binding Buffer (Ca2+). Add 5-10 ul of Annexin V-FITC and 5-10 ul of PI (100 ug/ml) Incubate at room temperature for 5-10 min in the dark. Analysis by FACS.

99 Apoptosis 0 h 6 h 12 h PI Annexin V

100 Fluorescence Activated Cell Sorting

101

102 Purification of human NK cell subsets. Positive Negative 98.5 CD56bright CD CD56dim CD3

103 The End

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

neutral red photodetector Andrew Moldavan 1960 Louis Kamentsky Moldavan microscope-based spectrophotometer 500 ultraviolet absorption scatter of blue Flow Cytometry flow cytometer DNA apoptosis ph flow cytometry flow cytometer (a) (b) cell counting instrument (c) 1960 ink-jet technology 17 19 1940 1950 fluorescence microscopy fluorescent dye DNA polyclonal