Introduction to Flow Cytometry -...

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Technology platform overview 1

Introduction to Flow Cytometry:

Analysis of 293 cells expressing GFP and RFP

Danièle Gagné M. Sc.Conseillère technique Cytométrie


What is Flow Cytometry?

• A powerful technique to analyze and characterize cells or particles

• A technology that simultaneously measures and then analyses multiple characteristics of single particles as they flow in a fluid stream through a beam of light

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Cell charaterization:

• Cell Lineage

• Subset identity

• Activation status

• Cell Cycle

• Chemokine Production

• Signal Transduction

• Migration/Homing Phenotype

Original from: Multicolor Flow Cytometry, Alan Stall, Vancouver 2005, BD Biosciences


To do Flow Cytometry:

• What you need?– Individual particles in suspension– Cells, beads, yeasts, bacteria, capsids, water/oil/water immersions …

• What you want?– Characterize each particle by measuring one or many parameters at the

same time– Separate desired particles from the bulk (in sorting mode)

• What you get?– Datas for every particle, for every parameter measured– Statistics for different subsets of particles (%, MFI, SD, CV)– Isolated particles (in sorting mode)

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How does it work?

Fluorescence

Detector A, B, C, etc…

Collection Lens /

Filters

FSCLaser


How does it work?

• Cells are transported from a random three-dimensional sample suspension to an orderly stream of particles traveling past one or more illuminating beams

• Illumination of stained and unstained cells by laser light; production of scatter and fluorescent light signals

• Light signals separated by optical filters according to their wavelenghts and captured by detectors

• Detected light converted to electronic signals, transmission to the computer for processing and data storage

• Datas can be reanalysed by specialysed softwares

Fluidic

Optic

Electronic

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Fluidic System

• Cells in suspension flow in a stream and pass one by one in front of a laser beam (interrogation point)

• Pressurized system using laminar flow

Sample injected into a stream of sheath fluid

The design of the « FlowCell » causes a hydrodynamic focusing of the samplein the center of the sheath fluid

Cells are aligned, one after the other


Fluidic System• Sample pressure (P) and sheath fluid pressure are different

• P sample > P sheath ; P sample = width of the sample core

low P high P

Original from: BD FACSAria User’s Guide, BD Biosciences, San Jose, 2003

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Optical System

• Generation and capturing of light signals

• <Excitation> Part :– Lasers

– Filters and mirrors to focus the laser beam to the flowcell and the fluidic core

• <Collection> Part :– Optical fibers to direct emitted light to the

appropriate set of detectors

– Filters to split the emitted light, according to the wavelenght, and direct it to a PMT


Optical System


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FSC

Detector

Collection

Lens

Laser Beam

Fluorescence

Detector A, B, C, etc…

Original from Purdue University Cytometry Laboratories, Modified by R. Duggan

Optical System


LSR II

Canto II ♦

FACSAria (Hood)

FACSAria #2 ♦

♦

♦

Lasers


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Filters



Filter Combination

From: BD FACSCanto II Flow Cytometer reference Manual

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Electronic System

• Detectors collect light signals (photons)

• Electrical parts treat those signals (conversion into electrical current and voltage pulse)

• Voltage pulses converted to digital values and datas are displayed by the computer

• Digital values are recorded in <List Mode> files


Electronic System : Detectors

• 2 types of photodetectors in flow cytometry

– Photodiodes

• Less sensitive, used for strong signals (ex: FSC detector)

– Photomultiplier Tubes (PMTs)

• Much more sensitive, used to detect weaker signals generated by SSC and fluorescence

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Electronic System

PMT Voltage Input

150V-999V

Current out Photons in

Linear

Amplification

Log

Amplification

orVoltage

Time

DetectorPhotons converted

to no. of electrons

Original from Becton Dickinson Training manual, Modified by R. Duggan


Time

Original from: Flow Cytometry Basic Training <A look Inside the box>, R. Duggan, CHUG

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FSC

Detector

FSC

Detector

Time

Threshold

(eg. 52)

Threshold

(eg. 52)



Pulse Height

Pulse Width

Pulse Area

Time

Vo

ltag

e In

ten

sity

Pulse


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Data collected for each cell

• Relative size (Forward scatter : FSC)

• Relative granularity or intracellular complexity (Side scatter : SSC)

• Relative fluorescence intensity (one intensity for each fluorochrome present)


FSC and SSC properties

FSC

SSC

SSC

Forward Scatter (FSC) :

•Diffracted light (detected at 180o, off the axis of the incident laser

beam)

•Related to cell size (surface)

Side Scatter (SSC) :

•Refracted and reflected light (collected at 90o to the laser beam)

•Related to cell granularity or internal complexity

LASER

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FSC vs SSC Profile

FSC

SS

CLymphocytes

Monocytes

Granulocytes

RBCs, Debris,

Dead Cells


Fluorochromes frequently used


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Fluorochromes: Alexa Fluor

Original from: Alexa Fluor Dyes, Symply the Best and Brightest, Molecular Probes, Invitrogen


Fluorescent proteins

Original from: Improved monomeric red, orange and yellow fluorescent proteins derived from

Discosoma sp. Red fluorescent proteins, Shaner et al., Nature Biotechnology, vol.22, no12,

p.1567-1572, 2004.

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Fluorescence Profile

Event #Param 1

FSC

Param2

SSC

Param 3

FITC

Param 4

PE

Param 5

APC

1 100 500 10 650 4

2 110 505 700 700 6

3 90 480 720 670 10

4 95 490 15 720 15

0………10………100………1000…….10000

# c

ell

s 52%

Original from: Flow Cytometry Basic Training <A look Inside the box>, R. Duggan, CHUGFITC


FITC vs APC fluorescence

Antigen with low expression:

low nb of Abs

low nb of fluorescent molecules

lower intensity of fluorescence

Antigen with high expression:

high nb of Abs

high nb of fluorescent molecules

higher intensity of fluorescence

Fluorescence intensity (FITC)

Flu

ore

sce

nce

inte

nsity (

AP

C)

10%

40%

30%

20%

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Fluorescence intensity (FITC)

Flu

ore

sce

nce

inte

nsity

(AP

C) 10

%

40%

30%

20%

5% 53%

38% 4%

FITC vs APC fluorescence


DNA staining

• Hoechst (UV laser, viable cells)

• Dapi (UV laser)

• PI (Propidium iodide)

• Draq5 (Viable cells)

• 7-AAD (7-amino-actinomycin D )• VyBrant DyeCycle stains (Viable cells,

orange, green, violet)

• Sytox Green (Yeast)

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1. Identify cells and markers to study

2. Find the best combination of Abs/fluorochromes

3. Prepare appropriate control samples: • Unstained cells

• Cells + 1 fluorochrome (one tube/fluorochrome)

• FMO (Fluorescence Minus One)

• Isotype controls…

• Viability Stain (exclude dead cells: PI, 7-AAD)

4. Acquire data

5. Analyse data

Experiment planning


Compensation


FITC PE

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Compensation Matrix

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Before compensation After compensation


GFP and RFP: compensation!

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• Detection up to 10 parameters and 8 fluorochromes

• 3 lasers (488nm, 633nm and 405nm) (4-2-2 config.)

eg: FITC/PE/PE-Cy5/PE-Cy7 APC/APC-Cy7

Hoechst/BD Horizon V500

• Diva software

• 40-tubes carousel for automatic loading

• Multiparameter applications

• Trained users

Equipment: BD FACSCanto II flow cytometer


• immunophenotyping

• cell cycle analysis

• apoptosis study

• cell proliferation analysis

• intracellular cytokines analysis

• signal transduction pathways study (phospho-proteins)

• gene expression measurement with fluorescent proteins (GFP, YFP, etc.)

• intracellular Ca2+, intracellular pH measurement

• FRET

• ROS …

Applications

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Simple analysis…

: 0% : 85.7% : 2.8%

Negative Control Positive Control Sample Test

Results from Pierre Zindy


Fluorescent proteins/ Gene expression

GFP+ YFP+

GFP+ YFP-

GFP- YFP+

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Apoptosis

Original from: Annexin V-PE

apoptosis detection kit I, BD

Pharmingen Technical Data Sheet,

BD Biosciences

•Annexin V

•Caspases

•Apoptosis-associated proteins


Many fluorochromes…

Perez & Nolan, Nature Biotechnology, Vol 20, p155-162

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Cell cycle analysis (yeast)

DNA staining with Sytox Green / Analysis with FlowJo software

Results from Hery Ratsima


Cell cycle analysis (G0 vs G1 ; G2 vs M)

NupA10

CTL

Hoechst

p-H

3

Hoechst

Py

ron

in

Results from Sonia Cellot

G0 vs G1 G2 vs M

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More complex application!

5%

Results from Martin Audet

Biosensor for V2R Endocytosis assay

45%

An

ti-H

A

VenusA

nti-H

AVenus

Without AVP With AVP


Applications: Phospho-Proteins

lysisY Z

Phospho-

specific

Ab blot

1. Limited opportunity to view

variability

2. Limited statistics

3. Requires sorting of subsets

4. Requires large #s of cells (106)

0.1 1 10 100 1000

Flow

cytometry

1. Possible to observe heterogeneity

2. Considerably enhanced statistics

3. Can subset via surface markers to gain

access to rare cell types

4. Requires fewer cells (103 - 104)

5. Simultaneous detection of multiple post-

translational modifications within

heterogenic cell populations

1

110

1100 4671

115

25 50

7

227

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Applications: Phospho-Proteins

IL2 Treated

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

0

20

40

60

80

100

100

101

102

103

104

0

20

40

60

80

100

100

101

102

103

104

0

20

40

60

80

100

Alexa 647 anti-Stat5 (pY694)

CD3-/CD20+CD3+/CD20-CD3-/CD20-

CD3 PE

CD

20

Pe

rCP

-Cy5

.5

IFNα Treated

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

0

20

40

60

80

100

100

101

102

103

104

0

20

40

60

80

100

100

101

102

103

104

0

20

40

60

80

100

CD3 PE

CD

20

Pe

rCP

-Cy5

.5

Original from: Analysis of Protein Phosphorylation and Cellular Signalling Events by Flow

Cytometry, Robert Balderas, BD Biosciences.


Cytometric Bead Array (CBA)

Y

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

DETECTOR

ANTIBODIES

LYSATE, SERUM OR

SUPERNATE (50uL)BEADS

YY

YY

Y

YY

YY

Y

YY

YY

YY

YY

+

Y

Y

Y

YY Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

WASH

READ ON

ANALYZER



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Chemokine-I CBA Assay Standard



IL-8

RANTES

MIG

MCP-1

IP-10

0 pg/ml 40 pg/ml

IL-8

RANTES

MIG

MCP-1

IP-10

IL-8

RANTES

MIG

MCP-1

IP-10

40 pg/ml 625 pg/ml

Tissue Culture Supernatants (HICK-3/4) 1:2 dilution

IL-8

RANTES

MIG

MCP-1

IP-10


Equipment: BD FACSAria cell sorters

Detection up to 14 parameters

and 12 fluorochromes 3 lasers (488nm, 633nm and 407nm) Biological hood for infectious cell sorting One- to four-way sorting into tubes

(1ml, 5ml,15ml) Sorting directly into plates or on slides

(possibility of cloning) Sorting by operator only

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Cell sorting applications

• Isolation, Purification of cell subsets

• Homogeneous population for:

Transplantation

Cell culture

Western blot

RNA extraction

…


Cell Sorting

Tiré de:Integrating Cytomics and Proteomics, Tytus Bernas, Gérald Grégori, Eli K. Asem , J. Paul Robinson, MCP Papers in Press. Published on October 28, 2005.

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Cell Sorting


Cell sorting followed by proliferation analysis in vitro

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In vitro tracking of cell division using CFSE(carboxyfluoroscein diacetate succinimidyl ester)

3 2 1 0

Number of divisions:

3 2 1 0

Even

ts

CFSE

Adapted from:

Lyons and Parish, JIM, 171;131(1994)


Proliferation of WT vs SMAD3 KO CD4 naive cells

Results from: Jean-Sébastien Delisle

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Cell sorting application:Screening by FRET

A

CG

FP

2-M

AV

S

FRET

GFP2-MAXX + eYFP-MAXX

~45 % of the transfected cells

have a positive FRET signal.

FRET

GF

P2-c

DN

A

GFP2-cDNA library + eYFP-MAXX

~1 % of the transfected cells have a

positive FRET signal.

B

GF

P2

FRET

GFP2 + eYFP-MAXX

~0.2 % of the transfected cells

have a positive FRET signal.

D

eYFP

co

un

t

eYFP-MAXX stable cell line

eYFP

co

un

t

Untransfected 293T cells

Results from Martin Baril


Instrumentation

• Z2 Coulter Counter

• AutoMACS separator

• BD LSR II flow cytometer

• BD Canto II flow cytometer

• 2 BD FACSAria cell sorters

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Contact information

Platform manager: Danièle Gagné x8094

[email protected]

Assistant: Gaël Dulude x44683

[email protected]

Platform PI: Dr Claude Perreault [email protected]

Web site: www.iric.ca

mailto:[email protected]



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