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Flow cytometry training garvan

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Flow cytometry training garvan

  1. 1. MLC Flow Cytometry Facility Introduction To Flow Cytometry Basic training Rob Salomon Garvan Institute of Medical Research Darlinghurst NSW Flow Cytometry
  2. 2. What Is Flow Cytometry ?
  3. 3. What Is Flow Cytometry ? Measurement METRY
  4. 4. What Is Flow Cytometry ? Cells Measurement CYTO METRY
  5. 5. What Is Flow Cytometry ?Flow Cells MeasurementFlow CYTO METRY
  6. 6. What Is Flow Cytometry ?Flow Cells MeasurementFlow CYTO METRY Flow Cytometry
  7. 7. Why use Flow Cytometry ?• Rapid analysis ( 3k- 200k events/second)• Individual event analysis• Quantifiable results• Multiple parameter analysis• Statistical relevance
  8. 8. Why use Flow Cytometry• Statistical Relevance – As we increase our number of observations we also increase the ability to resolve smaller and smaller changes The smallest flow file will generally contain at least 5000 events. It is not unusual to obtain >10^6 events
  9. 9. Flow and Imaging Comparison Imaging Flow CytometryCells per field/sec) Approx 100 20, 000No. of parameter <6 <24Quantifiable Maybe (using complex Yes analysis tool) 18 bit resolution 12- 16 bit (< 65,536 (262,144 channels) channels)Ave number of 1,000 - >10, 000analysed cells 10 field of 100Anatomical localisation Yes no
  10. 10. Prerequisites for Flow Cytometry1. Cells in single cell suspension2. Fluorescent probes3. Cytometer The key to good result is good sample Preparation http://www.photobiology.info/Zimmer.html - from Roger Y.Tsien)
  11. 11. What does a Flow Cytometer do?Analyses light signals to determine: Phenotype and Function Cd3
  12. 12. Basics uses of Flow Cytometry ?• Phenotyping• Apoptosis and cell death• Cell cycle, cell divising and DNA synthesis• Transduction/transfection confirmation• Cell tracking• Small particle analysis• Functional analysis – calcium flux, gene expression, dye efflux, mitochondrial activity• Marine and microorganism identification
  13. 13. Flow Lab rules• No access without training• No unfixed human samples• No unfixed PC2 samples• Clean instrument after run• Top up Sheath and empty waste at end of run (waste has 100 ml bleach added to empty container after emptying)
  14. 14. Instruments availableAnalysers – Calibur ( 2 Laser) – Canto I (2 Laser) – Canto II (3 Laser) – LSRII CFI (5 Laser) – LSRII SORP (7 Laser)Sorters – Aria Operator assisted – InFlux
  15. 15. Calibur* 2 laser 488nm ( blue) 633nm (red) 8 parameters SSC & FSC 3 x blue laser 1 x red laser Event rate < 3, 000
  16. 16. Canto I 2 laser 488 nm ( blue) 633 nm (red) 8 parameters SSC & FSC 4 x blue laser 2 x red laser Event rate < 15, 000
  17. 17. Canto II 3 laser 488nm ( blue) 633 nm (red) 405nm (violet) 10 parameters SSC & FSC 4 x blue laser 2 x red laser 2 x violet Event rate < 10, 000
  18. 18. LSRII CFI 5 laser 488nm ( blue) 633 nm (red) 405nm (violet) 355nm (UV) 561 nm (YG) 10 parameters SSC & FSC 4 x blue laser 2 x red laser 2 x violetEvent rate < 20, 000
  19. 19. 7 laserLSRII SORP ** 488nm ( blue) 633 nm (red) 405nm (violet) 355nm (UV) 561 nm (YG) 532nm (G) 514nm (514) 22 parameters SSC & FSC& FSC PMT 4 x blue 3 x red 4 x violet 2 UV 2 YG 3G 2 514
  20. 20. Instrument Power
  21. 21. What’s inside a Flow Cytometer ?• Flow cytometers have 3 key systems – Fluidics – Optics – Electronics
  22. 22. Fluidics system Wet Sheath Flow Waste cart filter Cell1. Top up atstart of run
  23. 23. Fluidics system Wet Sheath Flow Waste cart filter Cell1. Top up at start 2. Check andof run remove air bubbles
  24. 24. Fluidics system Wet Sheath Flow Waste cart filter Cell1. Top up at start 2. Check and 3. Ensure flowof run remove air cell is free from bubbles air and blockages
  25. 25. Fluidics system Wet Sheath Flow Waste cart filter Cell1. Top up at start 2. Check and 3. Ensure flow 4. Ensure no airof run remove air cell is free from bubbles in line bubbles air and blockages and waste height doesn’t change
  26. 26. Sheath filter
  27. 27. Fluidics Prime Canto I, and Canto II1. Turn system on2. Remove air From Sheath Filter3. Perform software fluidics startup
  28. 28. Fluidics Prime Calibur1. Turn system on2. Remove air From Sheath Filter3. Pressurise sheath tank4. Prime 2 x ( no Tube)5. Run TDw for 1 min – or until no air in waste lines
  29. 29. Fluidics PrimeLSRII / LSRII SORP1. Turn system on2. Remove air From Sheath Filter3. Turn laser off (if possible)4. Prime 2 x ( no Tube)5. Run TDW for 1 min – or until no air in waste lines
  30. 30. Instrument Startup
  31. 31. Flow Cell Hydrodynamic Low focusing of sample to laser intercept - (interrogation point) Medium Legend Laser intercept High Core Stream
  32. 32. Sample flow rate control
  33. 33. Optics Sample laser Laser Emission Spectral Laser emission interaction at delivery collection separation flow cellEnsurelasers are on- softwareLSRII SORPORHardware
  34. 34. Optics Sample laser Laser Emission Spectral Laser emission interaction at delivery collection separation flow cellEnsure Ensure Clean Flowlasers are on cell- softwareLSRII SORPORHardware
  35. 35. Optics Sample laser Laser Emission Spectral Laser emission interaction at delivery collection separation flow cellEnsure Ensure Clean Flow Achieved by filterlasers are on Cell selection- softwareLSRII SORPORHardware
  36. 36. Optics
  37. 37. Optics
  38. 38. Optics• Allows the excitation and the collection of the emitted light Steering LASER mirrors emission Flow Cell - Steering interrogation mirrors point
  39. 39. Optics cont.. Fluorescent and SSC Detectors Signal Detection FSC is achieved by detector collecting emitted or scattered light
  40. 40. Optics cont.. B530 Detector – FITC GFP 530/30 488/10 SSC Fluorescent and Detector 506 LP SSC signals are collected at rightEmission angles to thefrom blue 575/26 excitation laserlaser are progressively B575 Detector picked off to – PE, PI 556 LP facilitate multiple fluorochrome use
  41. 41. Spectral Separation• Dichroic mirrors • LP (Long Pass) – allows light longer than nominated wavelength to pass • SP (short Pass) – allows light shorter than nominated wavelength to pass• Band Pass filters • Restrict the wavelength of light that is allowed to pass
  42. 42. Spectral Separation • Band Pass filters • Restrict the wavelength of light that is allowed to passCentre of Width ofbandpass bandpass
  43. 43. Using PMT arrays Channel Common fluorochro me B 780 PE CY7 B 670 PE CY5 PerCP B610 Dichroic only B575 PE B 530 FITC/ GFP 488/10 SSC
  44. 44. Understanding PMT arrays Dichroic ring Band Pass ring PMT ring
  45. 45. Understanding PMT arrays positi Wave on length A B C D E FA
  46. 46. Understanding PMT arrays positi Wave on length A >488nm B C D E FA
  47. 47. Understanding PMT arrays positi Wave on length A >488nm B B C D E FA
  48. 48. Understanding PMT arrays positi Wave on length A >488nm B B >735nm C D E FA
  49. 49. Understanding PMT arrays positi Wave on length C A >488nm B B >735nm C D E FA
  50. 50. Understanding PMT arrays positi Wave on length C A >488nm B B >735nm C 750- 810nm D E FA
  51. 51. Understanding PMT arrays pos Wave length itio n C A >488nm B B >735nm C 750-810nm D ED F A
  52. 52. Understanding PMT arrays pos Wave length itio n C A >488nm B B >735nm C 750-810nm D 488-735nm ED F A
  53. 53. Understanding PMT arrays pos Wave length itio n C A >488nmE B B >735nm C 750-810nm D 488-735nm ED F A
  54. 54. Understanding PMT arrays pos Wave length itio n C A >488nmE B B >735nm C 750-810nm D 488-735nm E 655-735nmD F A
  55. 55. Understanding PMT arrays pos Wave length itio nE C A >488nm B B >735nmF C 750-810nm D 488-735nm E 655-735nmD F A
  56. 56. Understanding PMT arrays pos Wave length itio nE C A >488nm B B >735nmF C 750-810nm D 488-735nm E 655-735nmD F 670-735nm A
  57. 57. Configuration Documents
  58. 58. Understanding the PMT electronic signal Detector or PMT Electron Cascade DigitisationLight andsignal processing Amplification Voltage http://sales.hamamatsu.com /assets/applications/ETD/p mt_handbook_complete.pdf
  59. 59. Affect of PMT voltageLow voltage Negative population
  60. 60. Affect of PMT voltageLow voltage Mid Voltage Negative Negative population population
  61. 61. Affect of PMT voltageLow voltage Mid Voltage High Voltage Negative Negative Negative population population population
  62. 62. Types of signal• Scatter light • FSC and SSC • Always the same wavelength as excitation source• Fluorescent light • Always longer than the excitation source
  63. 63. Understanding Scatter Signals• WBC discrimination FSC has some similarities to size SSC has some similarities to granularity and complexity
  64. 64. Fluorescent Signals• Fluorescence may be used in the detection of : – Protein, RNA and DNA – DNA synthesis – Dye efflux – Organelle Activity A cytometer can – Change in pH detected light from – Protein interactions any system you can design that – Cell movement and division utilises – etc fluorescence
  65. 65. Examples of fluorescent probe use
  66. 66. Understanding Fluoroscence The fluorescentExcited molecule is excited e- by the excitation state e- source (laser). This imparts energy to e- electrons in the e- molecule which inResting e- then released as Mechanism of the molecule relaxes. The Fluorscence energy is released as light.
  67. 67. How do I choose my Fluorochromes ?• Antibody availability• Function – i.e. Mcherry Vs GFP• Fluorochrome brightness• Excitation source• Emission filters• Other fluorochromes/ Signals present in my sample (spectral overlap)
  68. 68. Fluorochrome BrightnessProbe QYAF488 0.92R-Pe 0.82AF546 0.79AF594 0.66 Quantum yield :APC 0.68 Is a measure of theA647 0.33 relative brightness ofeGFP 0.6 the fluorochrome. IT is measured as:Azumi Green 0.74ZS Green 1 0.91http://en.wikipedia.org/wiki/Fluorophore
  69. 69. Fluorescent protein tablehttp://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf
  70. 70. Choosing your Fluorochromes spectral viewers http://www.bdbioscience s.com/research/multicolo r/spectrum_viewer/index. jsp http://www.invitrogen.co m/site/us/en/home/supp ort/Research- Tools/Fluorescence- SpectraViewer.htmlUse the
  71. 71. Choosing your Fluorochromes spectral viewers http://www.bdbioscience s.com/research/multicolo r/spectrum_viewer/index. jsp http://www.invitrogen.co m/site/us/en/home/supp ort/Research- Tools/Fluorescence- SpectraViewer.htmlUse the
  72. 72. Choosing your Fluorochromes spectral viewers http://www.bdbioscience s.com/research/multicolo r/spectrum_viewer/index. jsp http://www.invitrogen.co m/site/us/en/home/supp ort/Research- Tools/Fluorescence- SpectraViewer.htmlUse the
  73. 73. Choosing your Fluorochromes spectral viewers http://www.bdbioscience s.com/research/multicolo r/spectrum_viewer/index. jsp http://www.invitrogen.co m/site/us/en/home/supp ort/Research- Tools/Fluorescence- SpectraViewer.htmlUse the
  74. 74. Understanding Spectral Overlap Effect of spectral overlap - Instrument View 120% 100%Percentage of Signal 80% in Detector 60% 40% 20% Spectral overlap 0% B 530 B 585 occurs when PE 5% 87% fluorochromes FITC 95% 13% excited by the same lasers emit in similar ranges.
  75. 75. Compensation Signal from FITC bright Compensation Controls 120 Signal Strength 100 120% 80 60 100% 40 20 80%Axis Title 0 overlap B 530 B 585 60% FITC bright 100 13 40% overlap 20% FITC dull 0% 120 Signal Strength B 530 B 585 100 FITC 100% 13% 80 Compensation is PE 5% 100% 60 40 applied at the 20 0 single event B 530 B 585 FITC dull 50 6 level
  76. 76. Effect of Compensation Digital compensation doesn’t change the underlying data it just allows us toUncompensated Data interpret it
  77. 77. Effect of Compensation Digital compensation doesn’t change the underlying data it just allows us toCompensated Data interpret it
  78. 78. How many Fluorochromes can I use ?• Most flow = 1- 3 fluorochromes• Basic phenotyping panel = 6-8 fluorochromes• Complicated panels = 11-12 flourochromes• High end = 17 fluorochromesSeventeen-colour flow cytometry: unravelling the immune systemStephen P. Perfetto, Pratip K. Chattopadhyay & Mario Roederer
  79. 79. Impact of increasing Flourochromes • Data get dramatically more complexParameters 2 3 4 8 12 18 22Populations 22 23 24 28 212 218 222Populations 4 8 16 256 4,096 262,144 4,194,304With 3 12 24 48 768 12,288 786,432 12,582,912scatterpopulations Number of populations – assuming each fluorochromes gives rise to only a positive and negative population
  80. 80. How do I get more ?Analysis Cell Sorting Sorting See It Sort It
  81. 81. Contact Details• Rob Salomon – r.salomon@garvan.org.au – (02) 9295 8431• Bookings (David + Lachlan) – Flow@garvan.org.au – (02) 9295 8432• http://linkage.garvan.unsw.edu.au/Flow/index.html
  82. 82. Data Acquisition• BD FACSDiVa interface

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