Introduction to Flow Cytometry

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This slide show forms part of the Introduction to Flow Cytometry seminar help by The Garvan MLC Flow Cytometry Facility. The Garvan MLC Flow Cytometry Facility is part of the Garvan Institute of Medical Research and is located in Sydney NSW.

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Introduction to Flow Cytometry

  1. 1. MLC Flow Cytometry Facility Introduction To Flow Cytometry 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. 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)
  8. 8. What does a Flow Cytometer do?Analyses light signals to determine: Phenotype and Function Cd3
  9. 9. What’s inside a Flow Cytometer ?• Flow cytometers have 3 key systems – Fluidics – Optics – Electronics
  10. 10. Fluidics Delivery of Low sample to laser intercept (interrogation Medium point) Legend Laser intercept High Core Stream
  11. 11. Optics• Allow the excitation and the collection of the emitted light Steering LASER mirrors emission Flow Cell - Steering interrogation mirrors point
  12. 12. Optics cont.. Fluorescent and SSC Detectors Signal Detection FSC is achieved by detector collecting emitted or scattered light
  13. 13. 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
  14. 14. ElectronicsDetector or PMT Electron Cascade Digitisation and processing Amplification Voltage http://sales.hamamatsu.com /assets/applications/ETD/p mt_handbook_complete.pdf
  15. 15. What type of signals do we see with Flow ?• Scatter – Forward Scatter (FSC) • parallel or Perpendicular FSC – Side scatter (SSC)• Fluorescence – FITC , PE, APC, GFP, DAPI (plus lots lots more)
  16. 16. Understanding Scatter Signals• WBC discrimination FSC has some similarities to size SSC has some similarities to granularity and complexity
  17. 17. 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
  18. 18. Examples of fluorescent probe use 10 5Fluorescence 2 (CD4) <B670L_B-A>: CD8-PerCP55 104 3 10 0 0 103 104 105 <R780_A-A>: bTCR-APCAF750 Fluorescence 1 (βTCR)
  19. 19. 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.
  20. 20. 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)
  21. 21. 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
  22. 22. Fluorescent protein tablehttp://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf
  23. 23. 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
  24. 24. 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
  25. 25. 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
  26. 26. 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
  27. 27. 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.
  28. 28. 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
  29. 29. Effect of Compensation Digital compensation doesn’t change the underlying data it just allows us toUncompensated Data interpret it
  30. 30. Effect of Compensation Digital compensation doesn’t change the underlying data it just allows us toCompensated Data interpret it
  31. 31. 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
  32. 32. 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
  33. 33. Visualising Signal Data Dot Density plot plot Statistical measures are also used toContourplot identify Histogram changes
  34. 34. 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
  35. 35. Why use Flow Cytometry ?• Rapid analysis ( 3k- 200k events/second)• Individual event analysis• Quantifiable results• Multiple parameter analysis• Statistical relevance
  36. 36. 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
  37. 37. How do I get more ?Analysis Cell Sorting Sorting See It Sort It
  38. 38. Contact Details• Rob Salomon – r.salomon@garvan.org.au – (02) 9295 8431• Bookings (Nikki and David) – Flow@garvan.org.au – (02) 9295 8432• http://linkage.garvan.unsw.edu.au/Flow/index.html

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