Introduction to Flow Cytometry IGC Workshop

General Programme

  Time Tutorial Room Speaker

Day 1

9h30-10h45 Fundamentals of Flow Cytometry Ionians Rui Gardner

11h00-13h00 Module 1 – Group 1Module 2 – Group 2 Analyzer room (Hands-on)

Day 2

9h30-10h45 Multicolor Analysis Ionians Rui Gardner

11h00-13h00 Module 1 – Group 3Module 2 – Group 1 Analyzer room (Hands-on)

Day 3

9h30-10h45 Applications in Flow Cytometry Ionians Claudia Bispo

11h00-13h00 Module 1 – Group 2Module 2 – Group 3 Analyzer room (Hands-on)

Day 4

9h30-11h00 Cell Sorting Ionians Rui Gardner

11h15-13h00 FlowJo Basic Training Giordano Bruno  

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

What is Flow Cytometry?

Flow Cytometry uic

Fundamentals of Flow Cytometry

Rui GardnerIGC – April 2, 2013

Introduction to Flow Cytometry IGC Workshop

Overview

3

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

• Fluidics: Hydrodynamic focusing, Flow Rate...

• Optics: Light, fluorescence, emission and detection...

• Electronics: pulse, data acquisition...

• Analysis: Tips on Data handling

Resources

4

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

http://PracticalFlowCytometry.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

Tutorials:• http://www.invitrogen.com/site/us/en/home/support/Tutorials.html

Resources (Web Tools)

5

BD Biosciences:http://www.bdbiosciences.com/research/multicolor/tools/index.jsp

Resources (Web Tools)

6

Life Technologies:http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Cell-and-Tissue-Analysis-Technical-Resources.html

Resources (Web Tools)

7

eBioscience:http://www.ebioscience.com/resources.htm

Resources (Web Tools)

8

Bioledgend:http://www.biolegend.com/webtoolstab

What is Flow Cytometry?

9

Cytometry:Measurement of physical and chemical properties of cells.

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

Flow Cytometry is not equivalent to FACS: Fluorescence Activated Cell Sorting

What is Flow Cytometry used for?

Flow cytometry can be used to count large numbers of cells or particles based on size, internal compexity, phenotype, cellular  state, cell function, DNA content, gene expression, and to quantify these same cellular properties at a single-cell level.

Key Advantages of Flow Cytometry

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

• Detection of extremely rare populations

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

0.02%

Flow Cytometry  does not...• Locate where a certain component is within a cell• Measure distribution of cellular components• Measure cell morphology

Flow Cytometer in a nutshell

11

Cells in suspensionflow in single file through…

Fluidics

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

Optics

converted to digital valuesthat are processed and analyzed in a computer.

Electronics

Flow Cytometers

12

FACSscan (BD)FACSCalibur (BD)CyAn ADP (BC)LSR Fortessa (BD) FACSCanto (BD)EC800 (Sony-iCyt)Sony Spectral Analyzer (Sony)CyFlow Cube (Partec)MacsQuant (Miltenyi Biotec)Guava (Merck-Millipore)Gallios (BC)Accuri C6 (BD)Attune (Life Technologies)etc

MoFlo Legacy (BC)FACSAria (BD)MoFlo XDP (BC)MoFlo Astrios (BC)Influx (BD)FACSJazz (BD)FACS Vantage (BD)EPICS ALTRA (BC)Avalon (Propel Labs)Synergy sy3200 (sony-iCyt)SH800 (Sony)etc

Flow Cytometers

Analyzers High Speed Cell Sorters

Fluidics

Interrogation point

14

Simplified Schematics

SheathFlow

SheathFlow

SampleInjected

Laser

Flow Chamber

HydrodynamicFocusing

Flow Chamber in a BD FACSAria Cell Sorter Flow Chamber in a BD LSR Fortessa

SheathFlow

SheathFlow

SampleInjected

Laser

Flow Chamber

Flow Rate

16

More cells passper unit time

SheathFlow

SheathFlowSample

Flow

Laser

High Flow Rate

Less cells passper unit time

SheathFlow

SheathFlowSample

Flow

Laser

Low Flow Rate

CyAn ADP

LSR Fortessa

FACSCalibur

FACScan

Flow Rate

17

Low Flow Rate

Laser

Sheath

Sample

High Flow Rate

Laser

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

High Power

Lower Power

High Power

Lower Power

Samples should be run at the lowest flow rate (differential pressure) as possible.

If increasing flow rate has:• No impact on population distribution: Use high flow rate.• Changes population distribution: Use low flow rate and increase cellular concentration.

Typical jet-in air cytometer

1 5 10 50 100 50010001

5

10

50

100

500

1000

P re s s u re p s i

vms

Exposure time

18

v ~ 5-30m/s

Therefore exposure time ~ 10-7-10-5 s

𝐯=√ 2∆𝐏𝜌Cell velocity is proportional to Sheath Pressure

𝐯=√ 2∆𝐏𝜌 𝐹

Laser beam height 

~ 5-20 µm

Typically, in sorters, sheath pressure can be reduced to increase exposure time, and therefore sensitivity and resolution of acquisition.

Sheath pressure in Analyzers is usually fixed.

Optics

Excitation - Lasers

20

488 nm

Most common laser wavelengths available

442

Multiple Laser Systems

21

Laser Interceptsin Flow Cell

Spatially Separated Laser Beams

FACSAria III (BD Biosciences)

FACSAria III (BD Biosciences)

Time delay between lasers must be determined for each instrument for a given sheath pressure.

Changes in Sheath Pressure will impact measurements.

Emission - Light Scatter

22

Light is reflected towards all directions

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

Forward Scatter (FSC)

23

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

Side Scatter (SSC)

24

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

(2D) Scatter Plot

25

Optics(Fluorescence)

Fluorescence (1)

27

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-Emission Spectrum (2)

Emission maximaExcitation maxima

Each Fluorochrome has a specific Excitation and Emission Spectra

Excitation Spectrum (1)

30

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.

Fluorescence Charts

Fluorescence Spectra Viewers (Web Tools)

Spectra Viewers:• BD: http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/• Life Technologies: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/

Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html• eBioscience: http://www.ebioscience.com/resources/fluorplan-spectra-viewer.htm• Bioledgend: http://www.biolegend.com/spectraanalyzer• UArizona: http://www.mcb.arizona.edu/ipc/fret/• Evrogen: http://www.evrogen.com/spectra-viewer/viewer.shtml

34

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

37

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

http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE - PLATE - Beach.jpg

Roger Tsien, Nobel Lectures, 2009

Tsien Lab

What is Flow Cytometry?

Flow Cytometry uic

Introduction to Flow Cytometry IGC Workshop

Coffee Break…!