Daisy KuoAssistant Product ManagerE-mail: daisy_kuo@bd.comBDBiosciences

Introduction to Flow Cytometry

-- BD FACSCanto IITM

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Outline

• Basic Concept of Flow Cytometry

• FACSCanto II System Introduction

• Application Examples

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What is Flow Cytometry?

• Flow = Fluid• Cyto = Cell• Metry = Measurement

• A variety of measurements are made on cells, cell organelles, and other objects suspended in a liquid and flowing at rates of several thousands per second through a flow chamber.

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Particle Size

• Detection range: 0.5~50um

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What Can a Flow Cytometer Tell Us About a Cell?

Its relative size (Forward Scatter—FSC)

Its relative granularity or internal complexity (Side Scatter—SSC)

Its relative fluorescence intensity

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Scatter Light

FSC Sensor

Laser

SSC Sensor

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Lysed Whole Blood

Forward Scatter

Sid

e S

catte

r

Lymphocytes

Monocytes

Neutrophils

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Fluorescence Light

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Fluorescence

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BD FACSCanto IITM

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Subsystems

FluidicsTo introduce and focus the cells for interrogation.

OpticsTo generate and collect the light signals.

ElectronicsTo convert the optical signals to proportional digital signals, process the signals, and communicate with the computer.

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sheath sheathsample

Sample Flow

Hydrodynamic Focusing

Excitation Lasers

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Sample Differential

Sample

High Sample Pressure

120 µL/min

High Differential Pressure

LaminarFlow

Sheath Sheath

LaminarFlow

Low Differential Pressure

Sheath SheathSample

Low Sample Pressure

12 µL/min

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Optics

• Excitation optics– Lasers Lenses to shape and focus the laser beam

• Collection optics– A collection lens to collect light emitted from the

article-laser beam interaction– A system of optical mirrors and filters to route

specified wavelengths of emitted light to designated optical detectors

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Fluorochrome Spectra

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Excitation Optics

• Spatially separated laser beams lower the possibility of fluorescence spillover

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Collection Optics

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Optics-- ConfigurationOther Fluorochrome

GFP

PI

PI, PE-Cy5.5, 7-AAD

Alexa Fluor® 633

DAPI, Hoechst Dye

Cascade Blue®

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PMTs and preamps convert photons to voltage pulses.

Analog-to-digital converters translate analog signals to proportional digital signals.

Compute area and height for each pulse.

Perform compensation and calculate ratios and width.

An embedded computer interfaces with the computer workstation for data transfer.

Electronics

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Creation of a Voltage Pulse

Laser

Time (µs)

Volts

Pul

se H

eigh

t

Pulse Width

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Analog-to-Digital Converter

Baseline

16,364

Time

937

1,985

7,650

12,420

Hei

ght

351

383

375

406

377

318

367

319

375

423

432

937

1985

7650

1242

015

300

1325

657

9124

7184

243

333

131

130

837

634

941

482

313

7390

351

433

841

830

731

735

331

370

340

337

830

840

640

530

335

340

532

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Digitized values

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Quantification of a Voltage Pulse

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Doublet Discrimination

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39,27139,271

Data Storage

Event 1Event 2Event 3

FSC SSC FITC PE0 60 120 89

10 160 65

30 650 160

List-Mode Data

39,271

89

675 30,621PE

FITC

675Time

PEFI

TC

39,271

22,688

30,621

6,189

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Data Display: Linear vs Log

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Spectral Overlap– Compensation Theory

APC PerCPPI

Wavelength (nm)400 500 600 700

100%

0%

Pacific Blue

Nor

mal

ized

Inte

nsity

FITC PEAmCyan PerCP-Cy5.5

800

PE-Cy7

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Spillover

FITC PE PerCP-Cy5.5

650 700

PerCP-Cy5.5695/40

500 600

FITC530/30

Rel

ativ

e In

tens

ity

Wavelength (nm)

550

PE585/42

PE-Cy7

PE-Cy7780/60

750 800

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FITC Spillover

650 700

PerCP-Cy5.5695/40

500 600

FITC530/30

Rel

ativ

e In

tens

ity

Wavelength (nm)

550

PE585/42

750 800

FITC

FITC

PEP

erC

P-C

y5.5

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650 700

PerCP-Cy5.5695/40

500 600

FITC530/30

Rel

ativ

e In

tens

ity

Wavelength (nm)550

PE585/42

FITC CompensationTo lower cluster, increase value.

FITC FITC

FITC

Per

CP

-Cy5

.5

FITC

Per

CP

-Cy5

.5

PE-%FITC

PerCP-Cy5.5-%FITC

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Compensation Examples

Correct Compensation Undercompensation Overcompensation

Incorrect Compensation

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Review

Time

Time

Time

Time

Time

Data Processing

PE

FITC

SS

C

FSC

AP

C

PerCP-Cy5.5

Time

ApplicationExamples

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Applications

• Phenotype Analysis (Cell Surface Antigens/Markers)• Intracellular Analysis

-- Eg. Cytokines, Signal Transduction molecules…etc.

• DNA Analysis-- Eg. Viability, Cell cycle, Apoptosis…etc.

• Cell Fuction Analysis-- Eg. Free radicals, Ca2+, Reporter genes…etc.

• CBA (Cytometric Bead Array)• Others

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• Ligand• Receptor• Adhesion molecule• …etc

Phenotype Analysis

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Lymphocyte Immunophenotyping

Granulocytes

Monocytes

Eosinophils

Basophils

NeutrophilsLymphocytes

T B NK

T Helper

TCytotoxic

Peripheral White Blood CellsCD45+

CD3+

CD4+ CD3+

CD8+

CD3+

CD3-

CD19+

CD3-

CD16+

CD56+

Monocytes

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Permeabilizing solution

• Cytokine• Enzyme• signal transduction

molecule• …etc.

Intracellular Analysis

Fixation solution

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Cytokine Detection

Picture From www.fredonia.edu

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CD3CD28

PHA

Con A

IonomycinLPS

T cell

Stimulation

Fixation

Secretion stop(Brefeldin A or Monensin)

Only in vitro

PermeabilisationIntracellular Staining

To enhance the accumulation of intracellular cytokines.

Monensin: Cytokines accumulate in the ERBrefeldin A: in Golgi complex.

To maintain structural integrity.Formaldehyde or glutaraldehydeKeep the protein structure and doesn't change the(accessibility of the) epitopes too much

Saponin (permeablisation buffer).

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Combination of Cell Surface and Cytoplasmic Staining

Th1/Th2/Th17 Phenotyping Kit

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Signal Transduction

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Intracellular Staining in Activated Lysed Whole Blood

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DNA Analysis

Detergent

Nucleic Acid Dye

Ethanol

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G2M G0

G1

s

0 200 400 600 800 1000

G0G1

s G2 M

DNA content

Count

2N 4N

Cell Cycle Analysis

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Apoptosis (Sub G1)

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• Membrane Potential (DiOC6, JC-1)

• Oxidative Metabolism (Free Radicals)

• Intracellular PH Value (Snarf-1)

• Ca++ Influx (Fluo-4/Fura Red, Indo-1)

• Phagocytosis

• Cell Proliferation (PI, BrdU, Intracellular Cyclins)

• Apoptosis (Annexin V, active Caspase-3)

Cell Function Analysis

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C a++ C a++C a++

C a++

Externalization ofphosphatidylserine

Plasmamembrane

Apoptosis

Cytoplasm Cytoplasm

Annexin V-FITCconjugate

Annexin V Assay

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Annexin V/PI Double Staining

Bordón et al. Radiation Oncology 2009 4:58

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Cytometric Beads Array (CBA)

Single StepIncubation

Two-StepIncubation

or

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Beads Provide a Flexible Platform

Multiple sizes

Different fluorescence

intensities

Different colors with different intensities

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Advantages of Bead-Based Immunoassays• Analyze multiple analytes simultaneously• Reduced sample volume requirements• Reduced hands-on time by parallel analysis of samples• Wide dynamic range of fluorescence detection (requires

fewer sample dilutions)

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Proteins MeasuredA. Interleukin (IL)-2B. IL-4C. IL-5D. IL-10E. Tumor Necrosis Factor-F. Interferon-

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Cytometry Beads Array (CBA)

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Standard Curves

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CBA Flex Sets• Open configuration (Up to 30 plex)• Clustering based on Red and NIR fluorescence intensity• Need to be used at dual-laser(488nm blue v.s 633nm red) instrument

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CBA Functional Beads• Can be conjugated with any Ab