Your Federal Quarterly Tax Payments are due April 15th Get Help Now >>

PowerPoint Presentation by eYhL5doH

VIEWS: 11 PAGES: 21

									Introduction To Flow Cytometry

             By
         Noha Kamel
 What is flow cytometry?



 Flow cytometry is a method of measuring multiple physical
 and chemical characteristics of particles by optical means.

• Peripheral blood, Bone marrow cells
• Bacteria
• Yeast
Flow Cytometry is a technology that simultaneously
measures and then analyzes multiple physical
characteristics of single particles, usually cells, as
they flow in a fluid stream through a beam of light.
 It measures and analyzes particles (cells) according
to:
 – Relative size
 – Relative granularity or internal complexity
 – Relative fluorescence intensity
• Flow cytometry integrates electronics,
  fluidics, computer, optics, software, and
  laser technologies in a single platform.
                     Fluorescence Activation Process
                        (or Immunofluorescence)
Antibodies recognize specific
                                             Antibodies are artificially
molecules in the surface of
                                             conjugated to fluorochromes
some cells
   FITC
              FITC          Antibodies




                     FITC                When the cells are analyzed by flow
             FITC
                                         cytometry the cells expressing the marker
                                         for which the antibody is specific will
                                         manifest fluorescence. Cells who lack the
                                         marker will not manifest fluorescence




   But not others
          Cellular Parameters Measured by Flow
  Intrinsic                               Extrinsic

• No reagents or probes required         • Reagents are required.
  (Structural)                              – Structural
    – Cell size(Forward Light Scatter)          • DNA content
    – Cytoplasmic grabularity(90                • DNA base ratios
      degree Light Scatter)                     • RNA content
                                            – Functional
                                                • Surface and intracellular
                                                  receptors.
                                                • DNA synthesis
                                                • DNA degradation (apoptosis)
                                                • Cytoplasmic Ca++
                                                • Gene expression
               Applications of Flow Cytometry.

• Cell size.
• Cytoplasmic granularity.
• Cell surface antigens (phenotyping).
• Apoptosis.
• Intracellular cytokine production.
• Intracellular signalling.
• Gene reporter.
• Cell cycle, DNA content, composition, synthesis.
• Bound and free calcium.
• Cell proliferation
• Cell sorting, single cell cloning
Phenotyping, Size and granularity detection:

               Sample requirements:

  A suspension of single cells or other particles
  in a suitable buffer, usually PBS.

  Typical density : 105 - 107 cells / ml




         +
                         Incubate                   Acquire
A cytometer consists of:




                               Electronics


Fluidics
                           Light detectors
                           FL3 PerCP, Cy5



                             FL2 PE
                                             Computer

                           FL1 FITC


                              SSC

 Laser
 Light                        FSC

 488nm
Size and Granularity




                       Size
            Light Scattering, 2 Parameter Histogram

                             Bigger
                               Apoptotic
                               Cells
90 degree                                        Bigger
Light Scatter                                    Cells
                              Dead
                              Cells                       More
   Y Axis
                                                          Granular




                       X Axis
                   Forward Light Scatter (FLS)            Live Cells
        Presenting and Interpreting Data

  Single parameters can be displayed as a histogram.




Dual parameter data can be displayed in two dimensions using
dot, density or contour plots.
                        1 Parameter Histogram

                                      Positive

               Negative


Count          Dimmer                              Brighter

        6


        4




        1


            1 2 3 4 6 7              150 160 170 .. 190
                    Channel Number
             Fluorescence picked up from the FITC
             PMT
               2 Parameter Histogram
 Single
 Positive PE                                         Double Positive
 Population                                          Population


PE FL




 Negative
 Population




               FITC FL             Single Positive
                                   FITC
                                   Population
                   Gating and Statistics

• Data generated in flow cytometry is displayed using
  Multiparamater Acquisition and Display software
  platforms.
• Histograms corresponding to each of the parameters of interest
  can be analyzed using statistical tools to calculate percentage
  of cells manifesting specific fluorescence, and fluorescence
  intensity.
• This information can be used to look at fluorescence
  expression within subpopulations of cells in a sample (gating).
                     Flow Cytometry Data

                           Smaller
                           Region,
                           Live cells
                           mostly




Larger Region
includes all cells
Gating:
Flow cytometry data analysis. Left-hand plot show a one-colour histogram plot
of CD8 expression by peripheral blood lymphocytes. Approximately 38% of
events fall between the marker boundaries, and are therefore regarded as CD8
+ve. The centre plot also shows CD8 expression on PB lymphocytes, but
depicts the relationship between CD8 and the T-cell marker CD3. The right-
hand plot shows a population of CD34 +ve 'stem cells' plotted against side
scatter.
Available analysis software

• Cellquest
• WinList
         Flow Cytometry : Reference Material

 Books
    • Practical flow Cytometry Howard M Shapiro.
    • Flow Cytometry: A Practical Approach MG Ormerod.
    • Introduction to Flow Cytometry, JV Watson.
    • Flow Cytometry: First Principals, Alice Givan.
    • Cytometric Analysis of Cell Phenotype and Function,
    McCarthy & Macey.



Journal
   • Cytometry : Journal of the International Society for
   Analytical Cytology. www.cytometry.org
         Flow Cytometry on the web


• ISAC (International Society For Analytical Cytology) www.isac-net.org
• Salk Institute http://flowcyt.salk.edu
• Purdue University www.cyto.purdue.edu
• Scripps Research Institute http://facs.scripps.edu/index.html
• Cancer Research UK http://science.cancerresearchuk.org/sci/facs

								
To top