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Flowcytometry: Principles, Applications and Data Analyses
Shahid Waseem, PhDCo-founder & MD
Alpha Genomics (Pvt) Ltd.
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• Principle/Introduction
• Instrumentation: Components/Working
• Applications
• Software
• Acquisition/Data Analysis
Out Line
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• Principle/Introduction
Out Line
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The analysis of single particles, often cells, within a heterogeneous population
• Whole blood, cell cultures, separated tissue, isolated nuclei, bacteria / yeast / parasites, algae & plankton etc.
• Signal from individual particles is collected for analysis as they pass through a laser in a stream of fluid
• Data displayed as events on histograms/dot plots etc.
Principle / What is flowcytometry?
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Multiple physical characteristics:
• particle’s relative size
• relative granularity or internal complexity
• relative fluorescence intensity
Principle / What is flowcytometry?
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• 488nm excitation:
• FITC, Alexa 4, GFP, YFP
• PE, PI, RFP,
• PerCP, 7-AAD, PE-Cy5*, PE-Cy7 (tandem dyes)
• 633nm excitation:
• APC, TOPRO-3, Cy5, Cy7
• Other Lasers:
• (488 nm, 638 nm, 405 nm, 375 nm, 561 nm, 808 nm)
Dyes
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FITC Compensation
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FITC Compensation
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• Instrumentation: Components/Working
Out Line
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Instrumentation: Components/Working
BD FACSCanto BD FACSAria II BD FACSCalibur
BD Accuri C6 BD FACScan
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Instrumentation: Components/Working
CytoFlex FC 500 Aquios
Gallios
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BD FACScan System
After completing this module, you will be able to:
• Describe the BD FACScan™ system and how it works
• Perform instrument startup and shutdown procedures
• Describe instrument maintenance procedures
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BD FACScan System
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BD FACScan System
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BD FACScan System
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BD FACScan System
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BD FACScan System
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BD FACScan System
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BD FACScan System
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• Applications
Out Line
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Applications
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Multicolour analysis
•Immunophenotyping
•Cells surface antigen detection (e.g. receptors, adhesion molecules)
•Intracellular staining
•Assessing infection/transfection levels
•Antibodies/ dyes/ Quantum dots
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Immunophenotyping
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Immunophenotyping
• CD34+ stem cell enumeration
• Method of repopulating stem cells following
radiotherapy treatment
• Patient treated to produce excessive levels of
pluripotent cells which are harvested from
peripheral blood
• Number of cells reintroduced, important in
success rate of procedure
• Abs vs stem cell markers CD34 and CD45
used in enumeration procedure
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Immunophenotyping (Cell Separation)
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Cell cycle analysis
DNA probes
DAPIHoechst UV
Propidium iodide (PI)7-AAD 488
TOPRO-3DRAQ5 633
These dyes are stoichiometric – number of bound molecules are equivalent to the number of DNA molecules present
Note the cell volume (size) and DNA
concentration change as the cell progresses
through the cell cycle
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Cell cycle analysis
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Cell cycle analysis
Measuring height against width gives us area
Two G1 cells together will have same PI intensity as a G2cell, but the area (signal h x w) will be greater andtherefore can be discriminated on a plot of signal W vs A
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Cell cycle analysis
•Bromodeoxyuridine (BrdU) incorporation
•A limitation to standard single colour DNA staining is that we can’tdetermine whether S-phase cells are actually cycling
•Cells take up BrdU during S-phase, but not during G1 or G2, anAb/PI vs BrdU then allows us to determine which cells are activelycycling within a population by two-colour analysis:
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Cell cycle analysis
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Cell cycle analysis
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Cell proliferation (CFSE)
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Apoptosis and Cell Viability
• Gene directed cell death•Cancer cells develop a strategy to evade apoptosis
Apoptosis can be analysed by FACS:
• Fragmentation of DNA (subG1 assay, Hoechst dyes)
• Membrane structure and integrity (AnnexinV, PI)
• Mitochondrial function (Mitotracker Red)
• Caspase activity (antibodies assay)
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Apoptosis and Cell Viability
DNA fragmentation allows apoptosis to be quickly assessed with eg. PI can be seen as apopulation of small peaks to the left of G1 in a histogram. Quick and easy way to determineif apoptosis is occurring
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Apoptosis (dye uptake method)
Changes in membrane permeability due to apoptosis allow intracellular dyes to stain unfixed cells
7-AAD (DNA)
Live cells exclude dye
Apoptotic cells stain 7-AADdim
Dead cells stain 7-AADbright
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Apoptosis/Necrosis (Annexin-V/PI)
Apoptosis
Necrosis
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• Software (CellQuest)
• Acquisition/Data Analysis
Out Line
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CellQuest
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CellQuest
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CellQuest
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CellQuest
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CellQuest
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CellQuest
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CellQuest
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CellQuest
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CellQuest
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CellQuest
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CellQuest (Acquisition)
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CellQuest (Stats)
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CellQuest (Formatting)
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CellQuest (Gating)
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Summary
• Flow cytometry is a powerful method for rapidly quantitating cellular fluorescence
• A number of functional assays such as cell cycle and apoptosis can be determined by flow and can be used as a method for assessing e.g. the effects of drugs on cell function, or the expression of mutant proteins
• Finally, cells and sub-cellular particles can be sorted from heterogeneous samples to yield near homogeneous populations for subsequent culturing or analysis.