Theory and Applications for Cell Sorter

Transcription

1 Theory and Applications for Cell Sorter Tony Liu 劉聖德 Product Manager BD Biosciences Taiwan Sep. 07,

2 Outline Basic Concept of Flow Cytometry FACSAria II System Introduction Cell Sorting Theory Application Examples Sorting Tips BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company.

3 Basic Concept of Flow Cytometry BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company.

4 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 liquidand flowing at rates of several thousands per secondthrough a flow chamber.

5 Particle Size Detection range: 0.5~40µm

6 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

7 Scatter Light Laser FSC Sensor SSC Sensor

8 Lysed Whole Blood Side Scatter Neutrophils Forward Scatter Monocytes Lymphocytes

9 Fluorescence Light

10 Fluorescence

11 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, Ca 2+, Reporter genes etc. CBA (Cytometric Bead Array) Others

12 FACS: Fluorescence-Activated Cell Sorter Developed by Prof. Leonard Herzenberg, Stanford University, 1974

13 Principle of FACS The combination of 3 technologies: 1. Fluorescence detection 2. Blood cell counter 3. Ink-jet technology Drop Charging Drop generation + + +

14 Evolution of FACS Technology BD FACS Museum, San Jose FACS II, 1976 FACS III, 1977 FACS IV, 1978 FACStar, 1984 FACVantage, 1992

15 FACSAria II System Introduction 2016 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company.

16 BD FACSAriaII Cell Sorter +

17 Main Components Fluidics To introduce and focus the cells for interrogation. Optics To generate and collect the light signals. Electronics To convert the optical signals to proportional digital signals, process the signals, and communicate with the computer. 17

18 Fluidics Cart Pressurized sheath tank (10L) Level sensor allows use of 95% of tank volume Pressurized ethanol shutdown tank (5L)

19 Sample Input

20 Sample Injection Chamber Temperature control (4, 20, 37, 42 C) Software-controlled agitation (100, 200, 300rpm) Automatic and user controlled sample line backflush

21 Sample Path

22 Sample Flow: Hydrodynamic Focusing sheath flow sheath flow sample flow laser beams

23 Optics Excitation optics Lasers Lenses to shape and focus the laser beam Collection optics A collection lens to collect light emitted from the article-laser beam interaction A system of optical mirrors and filters to route specified wavelengths of emitted light to designated optical detectors 23

24 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.

25 Excitation Optics Laser Wavelength Commonly Used Fluorochromes (nm) or Dyes Blue 488 nm FITC, PE, PE-TxRed, PE-Cy7, PerCP, PerCP-Cy5.5, PI Red 633 nm APC, APC-Cy7, APC-H7 Near UV 375 nm Hoechst, DAPI Upgraded

26 Collection Optics 26

27 Collection Optics: Optical Filters Longpass Bandpass LP 500 BP500/50 27

28 Collection Optics Octagon and Trigons NUV

29 Configuration (375-nm NUV laser) LP

30 Electronics Converts analog signals to proportional digital signals Computes area and height for each pulse Performs compensation, calculates ratios, and calculates width Interfaces with the computer for data transfer Performs sorting decisions, including gating, conflict resolution, and drop charging 30

31 Creation of a Voltage Pulse Laser Volts Pulse Height Pulse Width Time (µs) 31

32 Quantification of a Voltage Pulse

33 Data Storage Histogram (1 parameter) Event 1 Event 2 Event 3 Time List-Mode Data FSC SSC FITC PE , , ,271 30,621 22,6888 6,189 Dot Plot (2 parameters) 39,271 Dot plot (2 parameters) FITC FITC 89 PE ,621 PE

34 Data Display Linear Scaling Log Scaling

35 Linear v.slog Voltage Pulses Volts 0.1 V Time Volts 0.4 V Time Volts 1.2 V Time Volts 4 V Time Volts 8 V Time

36 Linear v. Log Amplification Linearamplification is usually used for light scatter parameters and DNA analysis. Logamplification is used for fluorescence signals with a large dynamic range.

37 Compensation Theory Emission Spectra: Spectral Overlap 100% Pacific Blue AmCyan FITC PE PI APC PerCP PerCP-Cy5.5 PE-Cy7 Normalized Intensity 0% Wavelength (nm)

38 Spillover FITC 530/30 PE 585/42 PerCP-Cy /40 PE-Cy7 780/60 Relative Intensity Wavelength (nm) FITC PE PerCP-Cy5.5 PE-Cy7 38

39 FITC Spillover FITC 530/30 PE 585/42 PerCP-Cy /40 Relative Intensity Wavelength (nm) 39

40 FITC Compensation FITC 530/30 PE 585/42 PerCP-Cy /40 Relative Intensity Wavelength (nm) Increase values 40

41 FITC Compensation To lower cluster, increase value. FITC 530/30 PE 585/42 PerCP-Cy /40 Relative Intensity Wavelength (nm) 41

42 Compensation Examples Incorrect Compensation Correct Compensation Undercompensation Overcompensation 42

43 Cell Sorting Theory 2016 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company.

44 Sort Gates 44

45 Sorting 2. Charge is applied via the streamcharging wire. 3. Charged droplets break off. 1. Sample generates light scatter and fluorescent signals; signals are analyzed. 4. Deflection plates attract or repel charged droplet. 5. Uncharged droplets pass to waste Charged drops containing particles of interest are collected.

46 Nozzles 70, 85, 100 or 130µm for different cell sizes (Cell size <= 1/6 Nozzle Size) Use different setting to generate drops

47 Drop Formation interrogation point breakoff

48 Drop Charging Time

49 interrogation point drop delay breakoff 49

50 Drop Delay BD FACS Accudrop technology Accudrop beads Diode laser Camera Optical filter drop delay interrogation point breakoff 50 Waste

51 Sweet Spot Automatic amplitude adjustment to keep the stability of drop formation -- Stream monitoring -- Clog detection 51

52 Sort Layout Devices 2 or 4 way Multiwell Plates Slides Custom Devices Precision mode Yield Purity Single cells 4-Way Purity Custom 52

53 Application Examples 2016 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company.

54 Sorting Applications on BD FACSAria II Cell Sorter Sorting cells with Live or Dead Morphology Specific surface marker profile High expression of transfected gene(s) Cell functions Cell physiology or metabolism Phases into cell cycle

55 Sorting by Morphology 10 to 14 µm Neutrophil 8 to 10 µm Lymphocyte 15 to 20 µm Monocyte

56 Sorting Viable Monocytes

57 Sorting for GFP + Cell Line Before Sorting After Sorting 6% 94%

58 Phenotype Analysis Ligand Receptor Adhesion molecule etc

59 Lymphocyte Immunophenotyping Peripheral White Blood Cells CD45 + Monocytes Monocytes Lymphocytes Granulocytes Neutrophils T B NK T Helper CD3 + CD3 - T Cytotoxic CD3 + CD4 + CD3 + CD8 + CD19 + CD3 - CD16 + CD56 + Basophils Eosinophils

60 Regulatory T Cells (Treg) Sorting CD4 + CD25 dim/+ CD127 -/dim

61 Sorting for Treg

62 Side Population (SP) Method

63 Background of SP First published by Goodell MA., 1996

64 Hoechst Excite best by UV laser Supravital minor groove-binding DNA stain with AT selectivity Uptake by live cells As DNA indicator on Flow Cytometry

65 Phenotype of SP Cells Efflux Hoechst dye via ABC (ATP-Binding Cassette) membrane pump Shows like an hook when displayed simultaneously at 2 emission wavelengths (Hoechst Blue/Red) + Verapamil (ABC inhibitor)

66 Tumor Tissue Analysis Single Cell Sorting Single Cell qpcr, PCR or WGS 1. Reveals intracellular differences in a heterogeneous population 2. Cell of interest are very difficult to obtain/isolate 3. Averaging effects of a pooled population

67 Single cell omics Very rapidly moving Tumor macrodissected into 12 sectors, and nuclei were isolated from six sectors and flow-sorted by ploidy. FACS profiles show four distributions of ploidy), which were gated to isolate 100 single cells. Navin et al 2011 Nature

68 Single cell workflow

69 Sorting Tips 2016 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company.

70 Sort Performance Depends on what you want: Purity Recovery Yield Viability Speed!!

71 Speed v.s Yield

72 Speed v.s Yield

73 Sample Preparation Considerations Enrich rare cell population if possible Avoid cell clumps Always filter your cells before sort! Use Accutase instead of Trypsin Treat cells with DNAse Cell concentration: PBMC: 1~2x10 7 /ml Cell line: 2~5x10 6 /ml Use appropriate sample buffer PBS, HBSS or phenol-red freeculture media w/ 25mM HEPES, 5mM EDTA and 1~2% FBS or 0.1~0.2% BSAto maintain cell viability Use viability dye to confirm cell viability before sort

74 Sorting Considerations Collection tube: Pre-coated with 1% BSA or 10% FBS overnight Filled with appropriate collection buffer (culture medium with 10~20%FBS) 5ml Falcon tube: 2ml 15ml centrifuge tube: 7ml Use Polypropylene instead of Polystyrene tubes Change collection tubes periodically to maintain cell viability Wash the sorted cells and resuspendin fresh culture medium In case of contamination, add 2X antibiotics in subsequent culture medium

75 75 Questions? 2016 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company.