Flowcytometry: Principles, Applications and Data...

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Flowcytometry: Principles, Applications and Data Analyses Shahid Waseem, PhD Co-founder & MD Alpha Genomics (Pvt) Ltd.

Transcript of Flowcytometry: Principles, Applications and Data...

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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

COMBINATION POPULATION IDENTIFIED

CD4+/CDw29+ Helper/effector, more mature memory cells

CD4+/CD45R+ Suppressor inducer, less mature non-memory cells

CD4+/Leu8+ Suppressor inducer, some helper function

CD4+/Class II MHC Activated cells, immature cells

CD4+/CD25+ Activated cells (IL2 receptor)

CD4+CD38+ Immature cells, activated cells

CD8+/CD11b+ of CD11b+ cells the suppressors are bright CD8+ and

NK are dim

CD8+/CD28+ Cytotoxic precursor/effector cells

CD8+/CD57+ Cytotoxic function

CD8+/Class II MHC+ Activated cells, immature cells

CD8+/CD25+ Activated cells (IL2 receptor)

CD8+/CD38+ Immature cells, activated cells

CD16+/CD57+ Low NK activity

CD16+/CD56+ Most potent NK activity

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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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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.