Flowcytometry: Principles, Applications and Data Analyses
Shahid Waseem, PhDCo-founder & MD
Alpha Genomics (Pvt) Ltd.
• Principle/Introduction
• Instrumentation: Components/Working
• Applications
• Software
• Acquisition/Data Analysis
Out Line
• Principle/Introduction
Out Line
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?
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
FITC Compensation
FITC Compensation
• Instrumentation: Components/Working
Out Line
Instrumentation: Components/Working
BD FACSCanto BD FACSAria II BD FACSCalibur
BD Accuri C6 BD FACScan
Instrumentation: Components/Working
CytoFlex FC 500 Aquios
Gallios
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
BD FACScan System
BD FACScan System
BD FACScan System
BD FACScan System
BD FACScan System
BD FACScan System
BD FACScan System
• Applications
Out Line
Applications
Multicolour analysis
•Immunophenotyping
•Cells surface antigen detection (e.g. receptors, adhesion molecules)
•Intracellular staining
•Assessing infection/transfection levels
•Antibodies/ dyes/ Quantum dots
Immunophenotyping
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
Immunophenotyping (Cell Separation)
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
Cell cycle analysis
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
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:
Cell cycle analysis
Cell cycle analysis
Cell proliferation (CFSE)
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)
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
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
Apoptosis/Necrosis (Annexin-V/PI)
Apoptosis
Necrosis
• Software (CellQuest)
• Acquisition/Data Analysis
Out Line
CellQuest
CellQuest
CellQuest
CellQuest
CellQuest
CellQuest
CellQuest
CellQuest
CellQuest
CellQuest
CellQuest (Acquisition)
CellQuest (Stats)
CellQuest (Formatting)
CellQuest (Gating)
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.