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Flow Cytometry

Basic Introduction and Applications

BD Biosciences

Grace Lin (grace_lin@bd.com)

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Outline

• Introduction to Flow Cytometry

• 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: 0.5~50µm

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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

ca

tte

r

Lymphocytes

Monocytes

Neutrophils

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Fluorescence Light

3

9

Fluorescence

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BD Flow Cytometers

FACSCantoII

2 Lasers, 6 Colors�細胞分析儀主體細胞分析儀主體細胞分析儀主體細胞分析儀主體�系統液流車系統液流車系統液流車系統液流車�BD FACStation電腦工作站電腦工作站電腦工作站電腦工作站

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Subsystems

Fluidics

To introduce and focus the cells for interrogation.

Optics

To generate and collect the light signals.

Electronics

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

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Fluidics

4

13

sheath sheath

sample

Sample Flow

Hydrodynamic Focusing

Excitation

Lasers

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Sample Differential

Sample

High Sample Pressure

60 µL/min

High Differential Pressure

LaminarFlow

Sheath Sheath

LaminarFlow

Low Differential Pressure

Sheath SheathSample

Low Sample Pressure

12 µL/min

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Fluorochrome Spectra

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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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Excitation Optics

• FACSCantoII--488nm Blue Laser--633nm Red Laser

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Optical Filters

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FACSCanto Collection Optics

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FACSCanto—Octagon and Trigons

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564–606 nm515–545

nm

750–810 nm

483–493 nm

Collection Optics—OctagonBlue Laser Signal

> 670 nm

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Collection Optics—TrigonRed Laser Signal

650–670 nm

750–810 nm

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FACSCanto Configuration

Wavelength PMT Position Intended Dyes Other Dyes(nm) (nm)

488 (blue) A PE-Cy7B PerCP, PerCP-Cy5.5 PI, PE-Cy5.5, DSRedC PE PI

D FITC GFPE Side Scatter (SSC)

633 (red) A APC-Cy7B APC Alexa Fluor® 633

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Electronics

• Convert analog signals to proportional digital signals.

• Compute area, height and width for each pulse.

• Interface with the computer for data transfer.

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Creation of a Voltage Pulse

Laser

Time (µs)

Vo

lts

Pu

lse

He

igh

t

Pulse Width

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Quantification of a Voltage Pulse

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39,27139,271

Data Storage

Event 1

Event 2

Event 3

FSC SSC FITC PE

0 60 120 89

10 160 65

30 650 16

List-Mode Data

675

Time

39,271

22,688

30,621

6,189

PE

FIT

C

Histogram (1 parameter)

39,271

89

675 30,621

PE

FIT

C

Dot Plot (2 parameters)

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Data Display

• Linear Scaling

• Log Scaling

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Linear v.s Log

Voltage Pulses

Time

Volts 8 V

Time

Volts 4 V

Time

Volts 1.2 V

Time

Volts 0.4 V

Time

Volts 0.1 V

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Linear v. Log Amplification

• Linear amplification is usually used for light scatter parameters and DNA analysis.

• Log amplification is used for fluorescence signals with a large dynamic range.

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Review

Time

Time

Time

Time

Time

Data Processing

PE

FITC

SS

C

FSC

AP

C

PerCP-Cy5.5

Time

Compensation Theory

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Emission Optics—Spectral Overlap

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Spillover

FITC PE PerCP-Cy5.5

650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC

530/30

Rela

tive I

nte

nsit

y

Wavelength (nm)

550nm

PE585/42

PE-Cy7

PE-Cy7780/60

750nm 800nm

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FITC Spillover

650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC

530/30

Rela

tive I

nte

nsit

y

Wavelength (nm)

550nm

PE585/42

750nm 800nm

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FITC Compensation

650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC530/30

Rela

tive I

nte

nsit

y

Wavelength (nm)

550nm

PE585/42

increase values

10

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650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC530/30

Rela

tive I

nte

nsit

y

Wavelength (nm)

550nm

PE585/42

FITC Compensation

To lower cluster, increase value.

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Compensation Examples

Correct Compensation Undercompensation Overcompensation

Incorrect Compensation

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Multicolor Analysis

Application Examples

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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)

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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 Cells

CD45+

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 ER

Brefeldin A: in Golgi complex.

To maintain structural integrity.

Formaldehyde or glutaraldehyde

Keep the protein structure and doesn't change the

(accessibility of the) epitopes too much

Saponin (permeablisation buffer).48

Combination of Cell Surface and

Cytoplasmic StainingTh1/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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G2

M

G0

G1

s

0 200 400 600 800 1000

G0G1

s G2 M

DNA content

C

o

u

n

t

2N 4N

Cell Cycle Analysis

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Sample Preparation

• Ethanol fixation-- Long term storage

• Detergent/Hypotonic cell permeabilization-- Better resolution-- Compatible with multicolor analysis if mild detergent is used

• Analysis in live cells-- For cell sorting

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DNA Dye Selection

• PI: The most popular DNA dye with high specificity; excited by 488nm blue laser

• 7-AAD: Alternative blue-excited dye for multicolor staining; less specificity

• DAPI: UV excitation with high specificity; for both

fixed and un-fixed cells

• Hoechst: UV excitation with high specificity; for

un-fixed cells

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Other Considerations

1. RNase Activity

2. Cells v.s PI Concentration Consistency

3. Sample Filtration (35um Filter)

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DDM (Doublet Discrimination)

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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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Mitochondria Membrane Potential (JC-1)

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Cytometric Beads Array (CBA)

Single Step

Incubation

Two-Step

Incubation

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 Measured

A. Interleukin (IL)-2

B. IL-4

C. IL-5

D. IL-10

E. Tumor Necrosis Factor-α

F. Interferon-γ

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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

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CBA Functional Beads• Can be conjugated with any Ab

Thank You!