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Experimental design Experimental design Niga Nawroly 1 Applications of Flow Cytometry

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Experimental design Experimental design Niga Nawroly 1 Applications of Flow Cytometry Powered By Docstoc
					Experimental design

     Niga Nawroly
                      1
Applications of Flow Cytometry.
• Cell size and cytoplasmic granularity/complexity.
• Cell surface antigens (phenotyping).
• Apoptosis.
• Intracellular proteins and cytokine production.
• Intracellular signalling, phosphorylation.
• Cell viability.
• Gene reporter (GFP, CFP, YFP….etc).
• Cell cycle, DNA content, composition, synthesis.
• Bound and free calcium.
• Cell proliferation (BRDU and CFSE).
• Cell sorting, single cell cloning (clonecyt).
  Design a “perfect experiment”


• What machine available?
• What laser/ filter configurations?
• Hence what fluorochromes / dyes can be used.
     Multi-colour flow cytometry
Advantages:
• Increase in information
• Saves time, sample and reagent
• Identify rare populations

Disadvantages:
• More complicated instrument setup
• Careful choice of fluorochrome combinations
• All reagents not available in all colours
• More controls required
Multi-colour flow cytometry
  experimental controls
            Experimental controls:

1.   Unstained cells
2.   Compensation tubes (single stains)
3.   Isotype control
4.   FMO control/ FMOPIC (FMO plus Isotype Control)
5.   Doublets discrimination
6.   Dead exclusion (live/dead)
7.   Other internal or external controls -scientific question
1. Unstained cells




                     7
             Unstained cells
   • Instrumental set up (Setting up voltages)
• In some experiments can act like a negative
  control




                                                 8
                  Unstained cells
Also,
• Can detect auto-florescence baseline.
• Unstained cells must have the same autofluorescence as your
  experimental sample




                                                            9
2. Compensation tubes (single stains)
                Compensation
Single stain controls (cells or beads)

Compensation Beads
• Better resolution, higher +ve %

Cells
• Resolution and % depend on the Ag
           Compensation
Cells of beads?

                          CD8+
                      Cells




                      Beads
           Compensation
Cells of beads?
                          Cells- cytokine




                              Beads
                       Compensation
        Why Compensation Beads?
• Cells too precious to ‘waste’ on compensation controls

• Compensating against the wrong background

• Ab of interest has low expression on cells

• Few positive cells present in sample

• Positive cells only present after stimulation or permeabilisation

•Minimise animal numbers
                  Compensation
       Why NOT Compensation Beads?

• Cells must be used for viability tests (SS for
live/dead)

• Cells must be used for GFP, CFP….. +ve

• Not for all fluorochromes and dyes
3. Isotype control
              Isotype control
immunofluorescence reagents used for measuring
 background fluorescence.

Same fluorochrome-conjugated antibody of an
  irrelevant specificity which has the same Ig
  isotype.
              Isotype control

• Isotype controls can be misleading by giving
  wrong information.

• They must be from the same manufacturer
  as the test antibody.

• Different F:P ratio.
         Isotype control
F:P ratio = 1:1
                       0.1%




F:P ratio = 3:1
                       5%
               Isotype control

• Exact same isotype

• Conjugation and F/P

• Concentration and titration

• Fluorochrome.


                                 20
     4.   FMO control/ FMOPIC
        (Fluorescence Minus One) /
Fluorescence Minus One plus Isotype control
        FMO control/ FMOPIC

An accurate way to identify positive events.
• FMO controls are necessary to identify cells
which do or do not express a given antigen.

• FMO controls should be used whenever
accurate discrimination is essential or when
antigen expression is relatively low (weakly
positive cell populations).
5. Doublet Discrimination




                            24
                Doublet Discrimination
The probability of doublets increases when :

1. Clumping cells analyzed.

2. The presence of high numbers of dead cells.

3. Flowing at high event rates.

4. Flowing at high pressure.
 To minimize doublet:
 • Proper sample preparation.
 • Pre-filtering of the sample.
                                                 25
 • Set up a doublet exclusion plot.
           Doublet Discrimination

Single cells
 Doublets
           FSC




                               SSC
                 Pulse width         FSC




                                           26
                          Doublet Discrimination




                                       FSc
                                                 Pulse width
CD117-APC




             No gate          Single cells   doublets            Clumps...
            (all cells)
                                                                             27
                                                  Bone marrow derived Mast cells
  Doublet Discrimination

Cell cycle analysis




                           28
6. Live/Dead Cell Discrimination




                                   29
             Dead discrimination
• Identification of non-viable cells in stained
  populations is essential for obtaining accurate
  data.

• Dead cells bind antibodies non-specifically. This
  could lead to misinterpretation.

• As a cell dies it's plasma membrane becomes
  permeable allowing fluorescent dyes present
  outside the cell to enter it and fluoresce.
                                                      30
            Dead discrimination
                    7AAD                  PI




             7AAD
SSC




                                 PI
      FSC
             SSC




                           FSC                       31
                             Bone marrow derived Mast cells
               Dead discrimination




                          FSC
         SSC




Dead cells
                 FSC
                                7AAD




                       7AAD
                                       32
              Dead discrimination




                              FSC
        SSC




Dead cells      FSC                  Invitrogen live/dead




                  Invitrogen live/dead


                                                            33
7. Other internal or external controls -
   scientific question
Other internal or external controls
        -scientific question
• The control is a particular sample that is
  treated the same as all the rest of the
  samples except that it is not exposed to
  manipulated variables -Treated vs untreated.

• Also other controls like fc receptor blocking.
              An Example
 Live/Dead                 Doublet discrimination




Lymphocytes         CD4+ T cells               Tregs
                                                    Tregs




                                     CD25
              CD3




                        CD4                   FOXP3
                Conclusion
• Include all the neseasry controls.

• Titrate your antibodies.


• Get help in designing
                       Thanks
•   Derek Davis
•   ISAC
•   Mario Roederer
•   Tamlyn Peel
•   Jens Loebbermann
•   Robert Sampson