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									Introduction to Flow Cytometry
                                                       IGC Workshop




                                                                                                           April 27-30, 2010


 General Programme

            Time              Tutorial                                     Room        Speaker
            9h30-10h45        Fundamentals of Flow Cytometry               Ionians     Rui Gardner (IGC)
 Day 1                        Policies and procedures when using the IGC
            11h00-13h00                                                    FACS room   Telma Lopes (IGC)
                              FACS equipment
            9h30-10h45        Multicolor Analysis                          Ionians     Rui Gardner (IGC)
 Day 2                        Module 1 – Group 1
            11h00-13h00                                                    FACS room
                              Module 2 – Group 2
            9h30-10h45        Applications in Flow Cytometry               Ionians     Rui Gardner (IMM)
 Day 3                        Module 1 – Group 2
            11h00-13h00                                                    FACS room
                              Module 2 – Group 3
            9h30-10h45        Cell Sorting                                 Ionians     Telma Lopes (IGC)
 Day 4                        Module 1 – Group 3
            11h00-13h00                                                    FACS room
                              Module 2 – Group 1

 Module 1 – Cell Cycle Analysis using FACScan (Telma Lopes)
 Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)
             What is Flow Cytometry?

Introduction to Flow Cytometry
                       IGC Workshop

         Flow Cytometry
                                      uic




          Fundamentals of Flow Cytometry



                                            IGC – April 27, 2010
                        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
                        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
                 What is Flow Cytometry?

   Cytometry:
           Measurement of physical and chemical properties of cells.

   Flow Cytometry:
            Characterization of cells flowing in a stream of fluid


                        Cells in suspension
Fluidics
                        flow in single file through
                        an illuminated volume where they
Optics                  scatter light and emit fluorescence
                        that is filtered, collected and
                        converted to digital values
Electronics
                        that are processed and analyzed in a computer


    FACS: Fluorescence Activated Cell Sorting

                                                                        5
      Key Advantages of Flow Cytometry




• Population data
    • Measure thousands of cells/particles per second
    • Measure multiple parameters simultaneously.


• Detection and sorting of extremely rare populations
                                                        0.02%

• Cell sorting
     • High purity, speed, and yield.
              Flow Cytometers




FACSCalibur                     FACSAria
FACSscan                        MoFlo
FACSCanto                       FACS Vantage
LSR II                          EPICS ALTRA
CyAn ADP                        etc
etc




                                               7
The Instrument




                 8
Interrogation point




                      10
Hydrodynamic Focusing



                  Low Flow Rate                      High Flow Rate

             Sheath            Sheath       Sheath               Sheath
              Flow    Sample    Flow         Flow       Sample    Flow
                       Flow                              Flow




     Laser                              Laser




                 Less cells pass                More cells pass
                 per unit time                   per unit time




                                                                          11
                                                        Flow Rate




                          Low Flow Rate                                                       High Flow Rate




                                         Sheath                                                               Sheath
         Laser




                                                                                Laser
                                 Sample
                                                                                                     Sample
                 3105




                 2328                                                   3105
Counts




                                                                        2328
                 1552
                                                         Counts




                                                                        1552


                 776
                                                                        776




                   0                                                      0
                                                                          100           101               102          103   104
                   100   101        102           103             104                                 FL2 Log Comp
                                FL2 Log Comp




                                                                                                                                   12
              Lasers



            488 nm




Most common laser wavelengths available




                                          14
Light Scatter




                Light is reflected towards all directions

                Low angle: Forward Scatter (FSC)
                High angle: Side Scatter (SSC)




                                                            16
Forward Scatter (FSC)



       The magnitude of Forward Scatter is roughly
           proportional to the size of the cell.




                                                     17
Side Scatter (SSC)




       Side Scatter is caused by granulosity and
         structural complexity inside the cell.




                                                   18
(2D) Scatter Plot




                    19
           Fluorescence (1)



Absorbed                      Excited State   High Energy
  Light



             Emitted
              Light



                                      Ground State
                                              Low Energy




                                                            21
    Fluorescence (2)



         2                         High Energy



                           2
1

                       1
                               3


     3
                                   Low Energy
      Excitation Spectrum (1)




          480 nm   520 nm   550 nm   590 nm




Excited




                                              23
Excitation-Emission Spectrum (1)


 Excitation




 Excitation max              Emission
    550 nm
Excitation - Emission




                  Excitation at different wavelengths
                   decreases intensity of emission.
Excitation-Emission Spectrum (2)



     Excitation maxima   Emission maxima
Fluorescence Charts
                        Fluorescence Spectra Viewers

Invitrogen (http:// www.invitrogen.com/spectraviewer)




                                                        BD Biosciences(http:// www.bdbiosciences.com/spectra)
                      Reactive and Conjugated Probes

Cascade Blue
Pacific Blue
Pacific Orange                                 Immunohistochemistry
Alexa Dyes
Lucifer yellow
NBD
R-Phycoerythrin (PE)
PE-Cy5 conjugates                     Direct method             Indirect method
PE-Cy7 conjugates
PE-Texas Red
PerCP
TruRed
PerCP-Cy5.5 conjugate
Fluorescein (FITC)
BODIPY-FL
TRITC
X-Rhodamine
XRITC
Lissamine Rhodamine B
Texas Red
Allophycocyanin (APC)
APC-Cy7 conjugates
Etc…




First dye-coupled antibody using fluorescein isothiocyanate
(FITC) patented by Joseph Burckhalter and Robert Seiwald,
in 1960


                                                                            Pictures adapted from wikipedia
Tandem Dyes


  PE-Alexa Fluor 700




PE-Cy5
PE-TxRed          PE-Cy7
PerCP-Cy5.5       APC-Cy7



                            31
                  DNA and Cell Function Probes



Calcium         Membrane       pH           Cell Tracker   DNA       Mitocondria
Fura 2          FM 1-43        SNAFLE       CMRA           DAPI      Nonyl Acridine O

Indo 1          FM 4-64        SNARF        CMTPX          HOECHST   MitoSox

Bapta/dye       DiO            BCECF        BMQC           TOPRO3    Mitotracker

Calcium Green   ANNINE 6       pHRodo       DMFDA          PI        Rhodamine 123

Fluo 4          Di4ANNEPS hq   lysosensor   CMTMR          DRAQ5     MitoProbe
             Fluorescent Proteins (1)




GFP was first noted by Shimomura et al., in the jelly fish Aequoria
Victoria, in 1962 and cloned 30 years later
Fluorescent Proteins (2)




                      San Diego beach scene drawn with living
                       bacteria expressing 8 different colors of
                                 fluorescent proteins
             What is Flow Cytometry?

Introduction to Flow Cytometry
                       IGC Workshop

         Flow Cytometry
                                      uic




                                       Coffee Break…!

								
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