Introduction to Flow Cytometry IGC Workshop

April 27-30, 2010

General Programme

Time Tutorial Room Speaker

Day 19h30-10h45 Fundamentals of Flow Cytometry Ionians Rui Gardner (IGC)

11h00-13h00 Policies and procedures when using the IGC FACS equipment FACS room Telma Lopes (IGC)

Day 29h30-10h45 Multicolor Analysis Ionians Rui Gardner (IGC)

11h00-13h00 Module 1 – Group 1Module 2 – Group 2 FACS room

Day 39h30-10h45 Applications in Flow Cytometry Ionians Rui Gardner (IMM)

11h00-13h00 Module 1 – Group 2Module 2 – Group 3 FACS room

Day 49h30-10h45 Cell Sorting Ionians Telma Lopes (IGC)

11h00-13h00 Module 1 – Group 3Module 2 – Group 1 FACS room

Module 1 – Cell Cycle Analysis using FACScan (Telma Lopes)Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)

What is Flow Cytometry?

Flow Cytometry uic

Fundamentals of Flow Cytometry

Rui GardnerIGC – April 27, 2010

Introduction to Flow Cytometry IGC Workshop

Overview

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• Definitions: What is flow cytometry and what does it measure?

• Fluidics: Hydrodynamic focusing

• Optics: Light, fluorescence, emission and detection

• Electronics: pulse, data acquisition

• Analysis: Data handling

Resources

Spectra Viewers:• http://www.bdbiosciences.com/spectra• http://probes.invitrogen.com/resources/spectraviewer• http://www.mcb.arizona.edu/ipc/fret/

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Tutorials:• http://probes.invitrogen.com/resources/education

Books:• Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version.

http://probes.invitrogen.com/lit/practicalflowcytometry/toc.htmlhttp://www.coulterflow.com/

Purdue Univ. Cytometry Labs: all you need to know about Cytometry!• http://www.cyto.purdue.edu

History of Flow Cytometry:• Shapiro, H. (2007) “Cytometry and Cytometers: Development and Growth”,

in Flow Cytometry with Plant Cells (Eds, J. Dolezel, J. Greilhuber, J. Suda),WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

What is Flow Cytometry?

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Cytometry:Measurement of physical and chemical properties of cells.

Flow Cytometry:Characterization of cells flowing in a stream of fluid

FluidicsCells in suspensionflow in single file through

Opticsan illuminated volume where theyscatter light and emit fluorescencethat is filtered, collected and

Electronicsconverted to digital valuesthat are processed and analyzed in a computer

FACS: Fluorescence Activated Cell Sorting

Key Advantages of Flow Cytometry

• Population data• Measure thousands of cells/particles per second• Measure multiple parameters simultaneously.

• Detection and sorting of extremely rare populations

• Cell sorting• High purity, speed, and yield.

0.02%

Flow Cytometers

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FACSCaliburFACSscanFACSCantoLSR IICyAn ADPetc

FACSAriaMoFloFACS VantageEPICS ALTRAetc

Flow Cytometers

Analyzers High Speed Cell Sorters

The Instrument

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Fluidics System

Interrogation point

10

Hydrodynamic Focusing

11

More cells passper unit time

Less cells passper unit time

SheathFlow

SheathFlowSample

Flow

Laser

High Flow Rate

SheathFlow

SheathFlowSample

Flow

Laser

Low Flow Rate

Flow Rate

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Low Flow Rate

Lase

r

Sheath

Sample

High Flow Rate

Lase

r

Sheath

Sample

100 101 102 103 104

FL2 Log Comp

0

776

1552

2328

3105

Counts

100 101 102 103 104

FL2 Log Comp

0

776

1552

2328

3105

Counts

100 101 102 103 104

FL2 Log Comp

0

776

1552

2328

3105

Counts

Optics(Lasers)

Lasers

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488 nm

Most common laser wavelengths available

Optics(Scatter Light)

Light Scatter

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Light is reflected towards all directions

Low angle: Forward Scatter (FSC)High angle: Side Scatter (SSC)

Forward Scatter (FSC)

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The magnitude of Forward Scatter is roughly proportional to the size of the cell.

Side Scatter (SSC)

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Side Scatter is caused by granulosity and structural complexity inside the cell.

(2D) Scatter Plot

19

Optics(Fluorescence)

Fluorescence (1)

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Fluorophore Energy Levels

Low Energy

High Energy

Ground State

AbsorbedLight

Excited State

EmittedLight

Absortion

StokesShift

Emission

Fluorescence (2)

Fluorophore

Absortion

StokesShift

Emission

Low Energy

High Energy

1

2

3

1

2

3

Lower Wavelength

Higher Frequency

Higher Energy

Higher Wavelength

Lower Frequency

Lower Energy

Excitation Spectrum (1)

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Excited

480 nm 520 nm 550 nm 590 nm

Excitation-Emission Spectrum (1)

Excitation max550 nm

Excitation

Emission

Excitation - Emission

Excitation at different wavelengthsdecreases intensity of emission.

Excitation-Emission Spectrum (2)

Emission maximaExcitation maxima

Fluorescence Charts

Fluorescence Spectra Viewers

Invitrogen (http:// www.invitrogen.com/spectraviewer)

BD Biosciences(http:// www.bdbiosciences.com/spectra)

Optics(Fluorescent Markers)

Reactive and Conjugated Probes

Cascade Blue Pacific Blue Pacific Orange Alexa DyesLucifer yellow NBD R-Phycoerythrin (PE) PE-Cy5 conjugates PE-Cy7 conjugates PE-Texas RedPerCP TruRed PerCP-Cy5.5 conjugateFluorescein (FITC)BODIPY-FL TRITC X-Rhodamine XRITCLissamine Rhodamine B Texas Red Allophycocyanin (APC) APC-Cy7 conjugatesEtc…

Immunohistochemistry

First dye-coupled antibody using fluorescein isothiocyanate (FITC) patented by Joseph Burckhalter and Robert Seiwald, in 1960

Direct method Indirect method

Pictures adapted from wikipedia

Tandem Dyes

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PE-Alexa Fluor 700

PE-Cy5PE-TxRedPerCP-Cy5.5

PE-Cy7APC-Cy7

DNA and Cell Function Probes

Calcium Membrane pH Cell Tracker DNA MitocondriaFura 2 FM 1-43 SNAFLE CMRA DAPI Nonyl Acridine O

Indo 1 FM 4-64 SNARF CMTPX HOECHST MitoSox

Bapta/dye DiO BCECF BMQC TOPRO3 Mitotracker

Calcium Green ANNINE 6 pHRodo DMFDA PI Rhodamine 123

Fluo 4 Di4ANNEPS hq lysosensor CMTMR DRAQ5 MitoProbe

Fluorescent Proteins (1)

GFP was first noted by Shimomura et al., in the jelly fish Aequoria Victoria, in 1962 and cloned 30 years later

Fluorescent Proteins (2)

San Diego beach scene drawn with living bacteria expressing 8 different colors of

fluorescent proteins

What is Flow Cytometry?

Flow Cytometry uic

Introduction to Flow Cytometry IGC Workshop

Coffee Break…!