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Introduction to Flow Cytometry

Introduction to Flow Cytometry. IGC Workshop. April 27-30, 2010. General Programme. Module 1 – Cell Cycle Analysis using FACScan ( Telma Lopes) Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner). What is Flow Cytometry ?. Flow Cytometry. uic.

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Introduction to Flow Cytometry

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  1. Introduction to FlowCytometry IGC Workshop April 27-30, 2010 General Programme Module 1 – Cell Cycle Analysis using FACScan (Telma Lopes) Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)

  2. WhatisFlowCytometry? Flow Cytometry uic Introduction to FlowCytometry IGC Workshop Fundamentals ofFlowCytometry Rui Gardner IGC – April 27, 2010

  3. Overview • Definitions: What is flow cytometry and what does it measure? • Fluidics: Hydrodynamic focusing • Optics: Light, fluorescence, emission and detection • Electronics: pulse, data acquisition • Analysis: Data handling 3

  4. Resources • Purdue Univ. Cytometry Labs: all you need to know about Cytometry! • http://www.cyto.purdue.edu • Tutorials: • http://probes.invitrogen.com/resources/education • Spectra Viewers: • http://www.bdbiosciences.com/spectra • http://probes.invitrogen.com/resources/spectraviewer • http://www.mcb.arizona.edu/ipc/fret/ • Books: • Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version. • http://probes.invitrogen.com/lit/practicalflowcytometry/toc.html • http://www.coulterflow.com/ • History of Flow Cytometry: • Shapiro, H. (2007) “Cytometry and Cytometers: Development and Growth”, • in Flow Cytometry with Plant Cells (Eds, J. Dolezel, J. Greilhuber, J. Suda), • WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4

  5. What is Flow Cytometry? Cytometry: Measurement of physical and chemical properties of cells. FlowCytometry: Characterization of cells flowing in a stream of fluid FACS: Fluorescence Activated Cell Sorting 5

  6. KeyAdvantagesofFlowCytometry • Population data • Measure thousands of cells/particles per second • Measure multiple parameterssimultaneously. • Detectionandsortingofextremelyrarepopulations • Cell sorting • High purity, speed, andyield. 0.02%

  7. Flow Cytometers Flow Cytometers Analyzers High Speed Cell Sorters FACSAria MoFlo FACS Vantage EPICS ALTRA etc FACSCalibur FACSscan FACSCanto LSR II CyAn ADP etc 7

  8. The Instrument 8

  9. Fluidics System

  10. Interrogation point 10

  11. Hydrodynamic Focusing High Flow Rate Low Flow Rate Sheath Flow Sheath Flow Sheath Flow Sheath Flow Sample Flow Sample Flow Laser Laser Less cells pass per unit time More cells pass per unit time 11

  12. Flow Rate High Flow Rate Low Flow Rate Sheath Sheath Laser Laser Sample Sample 12

  13. Optics(Lasers)

  14. Lasers 488 nm Most common laser wavelengths available 14

  15. Optics(Scatter Light)

  16. Light Scatter Light is reflected towards all directions Low angle: Forward Scatter (FSC) High angle: Side Scatter (SSC) 16

  17. Forward Scatter (FSC) The magnitude of Forward Scatter is roughly proportional to the size of the cell. 17

  18. Side Scatter (SSC) Side Scatter is caused by granulosity and structural complexity inside the cell. 18

  19. (2D) Scatter Plot 19

  20. Optics(Fluorescence)

  21. Fluorescence (1) Excited State High Energy Absorbed Light Stokes Shift Emitted Light Absortion Emission Ground State Low Energy Fluorophore Energy Levels 21

  22. Fluorescence(2) Stokes Shift High Energy 2 2 Absortion 1 1 3 Lower Wavelength Higher Wavelength 3 Low Energy Higher Frequency Lower Frequency Emission Higher Energy Lower Energy Fluorophore

  23. Excitation Spectrum (1) 480 nm 520 nm 550 nm 590 nm Excited 23

  24. Excitation-Emission Spectrum (1) Excitation Emission Excitation max 550 nm

  25. Excitation - Emission Excitation at different wavelengths decreases intensity of emission.

  26. Excitation-Emission Spectrum (2) Excitation maxima Emission maxima

  27. FluorescenceCharts

  28. FluorescenceSpectraViewers Invitrogen (http:// www.invitrogen.com/spectraviewer) BD Biosciences(http:// www.bdbiosciences.com/spectra)

  29. Optics(FluorescentMarkers)

  30. Reactive andConjugatedProbes CascadeBlue PacificBlue Pacific Orange AlexaDyes Luciferyellow NBD R-Phycoerythrin (PE) PE-Cy5 conjugates PE-Cy7 conjugates PE-TexasRed PerCP TruRed PerCP-Cy5.5 conjugate Fluorescein (FITC) BODIPY-FL TRITC X-Rhodamine XRITC LissamineRhodamine B Texas Red Allophycocyanin (APC) APC-Cy7 conjugates Etc… Immunohistochemistry Directmethod Indirectmethod Firstdye-coupledantibodyusingfluoresceinisothiocyanate (FITC) patentedbyJosephBurckhalterandRobertSeiwald, in 1960 Picturesadaptedfromwikipedia

  31. Tandem Dyes PE-Alexa Fluor 700 PE-Cy5 PE-TxRed PerCP-Cy5.5 PE-Cy7 APC-Cy7 31

  32. DNA andCellFunctionProbes

  33. FluorescentProteins(1) GFP was first noted by Shimomura et al., in the jelly fish Aequoria Victoria, in 1962 and cloned 30 years later

  34. FluorescentProteins(2) San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins

  35. WhatisFlowCytometry? Flow Cytometry uic Introduction to FlowCytometry IGC Workshop CoffeeBreak…!

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