Introduction to Flow Cytometry

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Daisy Kuo

Assistant Product Manager E-mail: [email protected] BDBiosciences

Introduction to Flow Cytometry

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Outline

• Basic Concept of Flow Cytometry

• Introduction to Instrument Subsystems

• 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

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Lysed Whole Blood

Forward Scatter Side Scatter Lymphocytes Monocytes Neutrophils 8

Fluorescence Light

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Fluorescence

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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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BD Flow Cytometers – Cell Analyzer

2 Lasers, 4 Colors Analyzer 2 Lasers, 6 Colors Analyzer FACSCalibur FACSCanto 12

BD Flow Cytometers – Cell Sorter

3 Lasers, 11 Colors Sorter

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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 — Pressurized System

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sheath sheath

sample

Flow Cell: Sample Flow

Hydrodynamic Focusing Excitation Lasers 16

Sample Differential

Sample High Sample Pressure 60 µL/min

High Differential Pressure

Laminar Flow

Sheath Sheath

Laminar Flow Low Differential Pressure

Sheath Sheath Sample Low Sample Pressure 12 µL/min 17

Fluorochrome Spectra

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Optics

• Excitation optics

– Lasers: 488nm(Blue) + 633nm(Red)  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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FACSCalibur Optics

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Optical Filters

21 488/10 FSC Diode FL3 670LP FL4 661/16 90/10 Beam Splitter DM 560SP DM 640LP Half Mirror SSC 488/10 585/42 FL2 FL1 530/30 Fluorescence Collection Lens

Collection Optics

Focusing Lens ~635 nm Red Diode Laser 488 nm Blue Laser Flow Cell

Excitation Optics

FACSCalibur Optics

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FACSCalibur Configuration

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FACSCanto Excitation Optics

488nm

633nm

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FACSCanto Collection Optics

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Detector Blocks—Octagon and Trigon

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Collection Optics—Octagon

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Collection Optics—Trigon

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Optics-- Configuration

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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) Vo lt s Pulse Height Pulse Width

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Quantification of a Voltage Pulse

32 39,271 39,271

Data Storage

Event 1 Event 2 Event 3 FSC SSC FITC PE 0 60 120 89 10 160 65 30 650 160 List-Mode Data 39,271 89 675 30,621 PE FITC 675 Time PE FITC 39,271 22,688 30,621 6,189 33

Data Display

• Linear Scaling

• Log Scaling

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Linear v.s Log

Voltage Pulses Time Vo lt s 8 V Time Vo lt s 4 V Time Vo lt s 1.2 V Time Vo lt s 0.4 V Time Vo lt s 0.1 V 35

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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Spectral Overlap– Compensation

Theory

APC PerCP PI Wavelength (nm) 400 500 600 700 100% 0% Pacific Blue Normalized Intensity FITC PE AmCyan PerCP-Cy5.5 800 PE-Cy7

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Spillover

FITC PE PerCP-Cy5.5 650 700 PerCP-Cy5.5 695/40 500 600 FITC 530/30 Relative Intensity Wavelength (nm) 550 PE 585/42 PE-Cy7 PE-Cy7 780/60 750 800 38

FITC Spillover

650 700 PerCP-Cy5.5 695/40 500 600 FITC 530/30 Relative Intensity Wavelength (nm) 550 PE 585/42 750 800 FITC FITC PE PerCP-Cy5. 5 39 650 700 PerCP-Cy5.5 695/40 500 600 FITC 530/30 Relative Intensity Wavelength (nm) 550 PE 585/42

FITC Compensation

To lower cluster, increase value.

FITC FITC FITC PerCP-Cy5.5 FITC PerCP-Cy5.5 PE-%FITC PerCP-Cy5.5-%FITC 40

Compensation Examples

Correct Compensation Undercompensation Overcompensation

Incorrect Compensation 41

Review

Time Time Time Time Time Data Processing PE FITC SSC FSC APC PerCP-Cy5.5 Time

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)

44 • Ligand • Receptor • Adhesion molecule • …etc

Phenotype Analysis

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Lymphocyte Immunophenotyping

Granulocytes Monocytes Eosinophils Basophils Neutrophils Lymphocytes T B NK T Helper _CytotoxicT

Peripheral White Blood Cells

CD45+ CD3+ CD4+ CD3 + CD8+ CD3+ CD3 -CD19+ CD3 -CD16+ CD56+ Monocytes 46 47 Permeabilizing solution • Cytokine • Enzyme • signal transduction molecule • …etc.

Intracellular Analysis

Fixation solution 48

Cytokine Detection

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49 CD3 CD28 PHA Con A Ionomycin LPS T cell Stimulation Fixation Secretion stop (Brefeldin A or Monensin) Only in vitro Permeabilisation Intracellular 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).

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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 54

G

₂

M

G

0

G

₁

s

0 200 400 600 800 1000

G

₀

G

₁

s

_G

2

M

DNA content C o u n t 2N 4N

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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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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 Step Incubation Two-Step Incubation or 60

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)

62 Proteins Measured A. Interleukin (IL)-2 B. IL-4 C. IL-5 D. IL-10

E. 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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