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

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• Principle/Introduction

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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?

Multiple physical characteristics:

• particle’s relative size

• relative granularity or internal complexity

• relative fluorescence intensity

Principle / What is flowcytometry?

• 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

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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

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Applications

Multicolour analysis

•Immunophenotyping

•Cells surface antigen detection (e.g. receptors, adhesion molecules)

•Intracellular staining

•Assessing infection/transfection levels

•Antibodies/ dyes/ Quantum dots

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

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

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

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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.