1. MLC Flow Cytometry Facility Introduction To Flow Cytometry Basic training Rob Salomon Garvan Institute of Medical Research Darlinghurst NSWFlow Cytometry

2. What Is Flow Cytometry ? 3. What Is Flow Cytometry ?MeasurementMETRY 4. What Is Flow Cytometry ? CellsMeasurement CYTO METRY 5. What Is Flow Cytometry ?FlowCellsMeasurementFlowCYTO METRY 6. What Is Flow Cytometry ?FlowCells MeasurementFlowCYTOMETRY Flow Cytometry 7. Why use Flow Cytometry ? Rapid analysis ( 3k- 200k events/second) Individual event analysis Quantifiable results Multiple parameter analysis Statistical relevance 8. Why use Flow Cytometry Statistical Relevance As we increase our number ofobservations we also increasethe ability to resolve smaller andsmaller changesThe smallest flow file will generally contain at least 5000 events. It is not unusual to obtain >10^6 events 9. Flow and Imaging ComparisonImagingFlow CytometryCells per field/sec)Approx 100 20, 000No. of parameter488nmBCDEFA 47. Understanding PMT arrayspositi Waveon lengthA>488nmB BCDEFA 48. Understanding PMT arrayspositi Waveon lengthA>488nmB B>735nmCDEFA 49. Understanding PMT arrayspositi Waveon lengthC A>488nmB B>735nmCDEFA 50. Understanding PMT arrayspositi Waveon lengthC A>488nmB B>735nmC750- 810nmDEFA 51. Understanding PMT arraysposWave lengthitionC A>488nmB B>735nmC750-810nmDEDFA 52. Understanding PMT arraysposWave lengthitionC A>488nmB B>735nmC750-810nmD488-735nmEDFA 53. Understanding PMT arraysposWave lengthitionC A>488nmEB B>735nmC750-810nmD488-735nmEDFA 54. Understanding PMT arraysposWave lengthitionC A>488nmEB B>735nmC750-810nmD488-735nmE655-735nmDFA 55. Understanding PMT arraysposWave lengthitionE C A>488nmB B>735nmF C750-810nmD488-735nmE655-735nmDFA 56. Understanding PMT arraysposWave lengthitionE C A>488nmB B>735nmF C750-810nmD488-735nmE655-735nmDF670-735nmA 57. Configuration Documents 58. Understanding the PMTelectronicsignal Detector or PMT Electron CascadeDigitisationLight andsignalprocessingAmplification Voltagehttp://sales.hamamatsu.com/assets/applications/ETD/pmt_handbook_complete.pdf 59. Affect of PMT voltageLow voltage Negative population 60. Affect of PMT voltageLow voltageMid Voltage Negative Negative population population 61. Affect of PMT voltageLow voltageMid Voltage High Voltage Negative Negative Negative population population population 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. Understanding Scatter Signals WBC discrimination FSC has some similarities to size SSC has some similarities to granularity and complexity 64. Fluorescent Signals Fluorescence may be used in thedetection of : Protein, RNA and DNA DNA synthesis Dye efflux Organelle ActivityA cytometer can Change in pH detected light from Protein interactionsany system youcan design that Cell movement and division utilises etc fluorescence 65. Examples of fluorescent probe use 66. Understanding FluoroscenceThe fluorescentExcited molecule is excited e- by the excitationstatee- source (laser). Thisimparts energy to e-electrons in thee-molecule which inRestinge- then released asMechanism of the molecule relaxes. TheFluorscenceenergy is releasedas light. 67. How do I choose myFluorochromes ? Antibody availability Function i.e. Mcherry Vs GFP Fluorochrome brightness Excitation source Emission filters Other fluorochromes/ Signals present in mysample (spectral overlap) 68. Fluorochrome BrightnessProbeQYAF488 0.92R-Pe0.82AF546 0.79AF594 0.66 Quantum yield :APC 0.68Is a measure of theA6470.33 relative brightness ofeGFP 0.6 the fluorochrome. ITis measured as:Azumi Green 0.74ZS Green 10.91http://en.wikipedia.org/wiki/Fluorophore 69. Fluorescent protein tablehttp://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf 70. Choosing your Fluorochromesspectralviewers 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. Choosing your Fluorochromesspectralviewers 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. Choosing your Fluorochromesspectralviewers 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. Choosing your Fluorochromesspectralviewers 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. Understanding SpectralOverlap Effect of spectral overlap - Instrument View 120% 100%Percentage of Signal 80%in Detector 60% 40% 20% Spectral overlap0%B 530 B 585occurs whenPE5%87% fluorochromesFITC 95%13%excited by thesame lasers emitin similar ranges. 75. Compensation Signal from FITC bright Compensation Controls120 Signal Strength100 120%80 60 100%40 20 80%Axis Title0 overlapB 530B 585 60% FITC bright 100 13 40% overlap 20% FITC dull0% 120 Signal Strength B 530 B 585 100FITC 100%13%80 Compensation isPE5% 100% 6040applied at the20 0 single eventB 530B 585FITC dull50 6level 76. Effect of CompensationDigital compensationdoesnt change the underlying data itjust allows us toUncompensated Data interpret it 77. Effect of CompensationDigital compensationdoesnt change the underlying data itjust allows us toCompensated Data interpret it 78. How many Fluorochromes can I use ? Most flow = 1- 3 fluorochromes Basic phenotyping panel = 6-8fluorochromes Complicated panels = 11-12flourochromes High end = 17 fluorochromesSeventeen-colour flow cytometry: unravelling the immune systemStephen P. Perfetto, Pratip K. Chattopadhyay & Mario Roederer 79. Impact of increasing Flourochromes Data get dramatically more complexParameters234 812 1822Populations 22 23 2428 212218 222Populations 48162564,096262,144 4,194,304With 312 24 4876812,288 786,432 12,582,912scatterpopulationsNumber of populations assuming each fluorochromes givesrise to only a positive and negative population 80. How do I get more ?Analysis Cell Sorting Sorting See It Sort It 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. Data Acquisition BD FACSDiVa interface