January 31, 2013 Ian Dimmick (Newcastle University) Graham ...docs.abcam.com/pdf/events/compensation-webinar-slides.pdfCD27 APC CD27 APC KLRg1-A488 KLRg1-A488 CD45ra PB CD27 APC KLRg1-A488

Colour Compensation in Flow Cytometry

January 31, 2013

Ian Dimmick (Newcastle University)

Graham Pockley (Nottingham Trent University)

Overview

Why do multicolour experiments?

Which Fluorochromes do I use?

What is spectral overlap and why is compensation needed?

2

compensation needed?

Procedure for compensation

Spill over examples

Spill over calculation procedure

Controls




3



Spill over examples


Controls

3Ab+3Ab=18 Phenotypes

2

1 4

5

4

1

3

4

6

2

3

5

6

18 Phenotypes in 2 tubes for 6 antisera

6Ab’s=36 Phenotypes

1

2

1

3

1

4

1

5

1

6

5

36 Phenotypes in 1 tube for 6 antisera

2

3

2

4

2

5

2

6 4

3

4

5

3

5

3

6

4

6

5

6



Which Fluorochomes do I use?

6



Spill over examples


Controls

When fluorochromes leave their excited state, they release energy in the form of a photon whose wavelength is longer that the excitation wavelength

7

Fluorescence Spectral viewers

8

Types of Fluorochromes(Non Tandem)

How bright is my Fluorochrome ?

FluorochromeAbs Emit

εεεεLaser FluorescenceQf

9

Specific wavelength(abs. Max)

Measured over entirespectra Values from 0.5-<1

Fluorescence intensity εεεε x QfTheoretical

Stain indices (SI=D/W)

Fluorochromea Stain index1PE-Cy5® 353

PE 3022APC 2782Alexa Fluor® 647 214

PE-Cy7® 1391PerCP-Cy5.5® 1073BD Horizon™ V450 85

10

“reserve the brightest fluorochromes for antigens with the lowest cellular concentration and vice versa”

3BD Horizon™ V450 853Pacific Blue™ 804Alexa Fluor® 488 73

Alexa Fluor® 700 614FITC 565APC-Cy7® 371PerCP 37

AmCyan 255APC-H7 24

Holden Maecker & Joe Trotter, Nature Methods 5, (2008)

How bright is the fluorochrome?

“Bright” = Good resolution sensitivity

Signal

Stain index

11

Signal

Cell

Types of Fluorochromes

Non Tandem

12

Tandem

Q dots

Non Tandem

13

Non Tandem

mean positive 57,382 mean positive 5,936

14

Compensation 525-450 14% Compensation 525-450 30.4%

15

Tandem dye breakdown

20mins RT 24 Hrs 4C 24 Hrs RT

16

20.98 22.75 34.59

Q Dots

17


What is spectral overlap and why is compensation needed?What is spectral overlap and why is compensation needed?


18



Spill over examples


Controls

is compensation needed?

What is spectral overlap?

520/20 585/42 660/20

What does spectral

overlap look like on

my plots?

compensation objectives

PE

PE FITCPE

FITC PE

19

V4 b

450nm 500nm 550nm 600nm 650nm 700nm 750nm 800nm

FITC

FITC → FITC →

PE

→

PE →FITC

FITC

PE

500nm 550nm 600nm 650nm

FITC PE

Slide courtesy of Dr Rebecca Stewart

Multicolour panels, addressing dye vector, and laser/filter configuration

20

The more PMTs the closer the filters, the higher the compensation values

Compensation is Fi independentIn

tensity o

f sig

nal

G Y R FR

Compensation set – on spectral overlap values

Despite the decrease in fluorescence

intensity, the spillover still represents

10% of the total fluorescence

21

Inte

nsity o

f sig

nal

Wavelength

G-Y Y-G Y-R R-Y FR-Y FR-RSpillover on yellow detector

represents 10% of toral fluorescenceAn increase in the green detector

sensitivity amplifies the amount of

signal collected but the spillover for

yellow remains constant;

compensation decreases

Increased

gain on

green

detector

Compensation

Dependant on PMT Voltages

22

Available Lasers

Fluorochromes

Instrument filter configuration




23


Spill over examples


Controls


FIT

vs

FITC

vs

PE

vs

FSC

vs


No. fluorochromes No. plots

2 1

Example: Three-colour experiment CD3-FITC , CD8-PE and CD19-PerCPCy5.5®

Step 1: Construct your acquisition panel

vs

PE

vs

PerCPCy5.5®

vs

PerCPCy5.5®

vs

SSC

FITC PE PerCPCy5.5®

24

HAVE ALL FLUOROCHROME COMBINATIONS IN YOUR PANEL!HAVE ALL FLUOROCHROME COMBINATIONS IN YOUR PANEL!

2 1

3 3

4 6

5 10

6 15

7 21

8 28

9 36

10 45

AK7

Slide 24

AK7 We don't need this slide, it's the same as the one afterAris Krikelis, 13/12/2012


Example: Three-colour experiment CD3-FITC CD8-PE CD19-PerCPCy5.5®

Step 3: Check for spectral overlap by running each fluorochrome separately and apply compensation if necessary

Tube 1: FITC only

Tube 2: PE only (remember to transfer your compensation values from your FITC tube!)

Look for the plots with PE on the axis: is there spectral overlap?

PE is spilling into FITC:

Adjust FITC -%PE

PE is spilling into PerCPCy5.5® :

Adjust PerCPCy5.5® -%PE

25

Look for the plots with PE on the axis: is there spectral overlap?

PE is spilling into FITC and PerCPCy5.5®


Example: Three-colour experiment CD3-FITC CD8-PE CD19-PerCPCy5.5®

Step 4: Run your stained cell sample

26




27




Controls

Spill over examples

Multicolour compensation Spill over

Where are my photons Qdot 605

28


Where are my photons PE

29


Where are my photons Pacific Blue™

30


Where are my photons PeCy5®

31




32



Spill over examples

Controls


Spectral overlap –calculation of Spill over

33

Compensation can be performed on cells if you have a bright signal, preferably as bright or brighter than anything you will encounter within your experiment


34

Compensation can be performed on dual cell populations if you have a bright signal for each antigen, preferably as bright or brighter than anything you will encounter within your experiment and the antibodies are against mutually exclusive antigens


Compensation beads

Polystyrene microparticles bind any kappa light chain-bearing immunoglobulin (mouse, rat, hamster and comp beads plus)

Kappa LCRantibody

35

Anti-Kappa

Each set of beads also contains negative beads with no binding capacity

Spectral overlap calculation of Spill over

AB. Fluorochrome combinations

The

Weak antigen : try to use a bright fluorochrome

Differentiate between compensation and data manipulation

If a fluorochrome is excited by more than one laser you

36

The Rules

are

If a fluorochrome is excited by more than one laser you need to evaluate if it is usable in a multicolour experiment

Always evaluate percentages of antigens by single colour controls, then check that the same percentage is achieved in your multicolor experiment

The lower the compensation values within your experiment, the more stable your experiment will be

Spectral overlap calculation of Spill over AB.

Fluorochrome combinations

Differentiate between compensation and data manipulation

37

Multiple laser excitation of fluorochromes

38

31%

9 colour well expressed antigen

39

10 colour S

ide s

catt

er

CD

4 P

e c

y7

CC

R7 P

e

CD

28 p

p 5

.5

CD

28 p

p 5

.5

CD3 Q605 CD8 APC-7 CD8 APC-7 CD27 APC CD27 APC

40

KLR

g1-A

488

PD

1-A

532

CD

57 A

350

CD

57 A

350

CD

57 A

350

CD

57 A

350

CD

4 P

e c

y7

CD

27 A

PC

A488A532Q605PercP5.5PeCy7APCAPC-Cy7Pac BlueA350A700

A488A532Q605PercP5.5PeCy7APCAPC-Cy7Pac BlueA350PE

CD27 APC CD27 APC KLRg1-A488 KLRg1-A488 CD45ra PB

CD27 APC KLRg1-A488 KLRg1-A488

CC

R7 P

eC

7

CD

4 A

700

Images provided by Jedrej Hoffmann, Ian Dimmick, Ioakim Spyridopoulos

Spectral overlap calculation of Spill over

AB. Fluorochrome combinations

Always evaluate percentages of antigens by single color controls, then

check that the same percentage is achieved in your multicolor experiment (Single [Single] Multicolour [M] ) diff%

S/M diff, %

A488 3.1

41

A532 0.3

Q605 3

PerCyp5.5 1.1

PeCy7 2.2

APC 3

A700 2.7

APC-CY7 2

Pac Blue 4

A350 1

Spectral overlap Calculation of Spill over

AB. Fluorochrome Combinations

High compensation

Values, increased

Compensation Stability

42

Instability,

Instrument Fi

monitoring critical low compensation

Values, decreased

Instability,

instrument Fi

monitoring less critical




43



Spill over examples


Controls

Controls

Gold standard

The negative cells that are clearly distinguishable from the positive population in a stained sample

Negative cells

Instrument noise, autofluorescence

Isotypic Same protein concentration / Ig isotype /fluorochrome as Ab

44

controlSame protein concentration / Ig isotype /fluorochrome as Ab

Isotypic control (Bad)

Different protein concentration/Ig isotype /fluorochrome manufacturer

Isoclonic control

The use of unconjugated test specific antibody to block specific antigen sites to leave only non specific binding sites available to the conjugated test specific antibody

Isotype controls

When to use and when not to use

Picking the correct Isotype control

45

Picking the correct Isotype control

F:P ratio

Protein concentration considerations

Test Antibody+++++++++++NS+

46

No Antibody

Isotype Ctl Ig subtype matched Protein conc. MatchedInevitably will have Different tert. ConfigThan test Ab

-

NS+

Fluorescence minus one FMO on weak antigens

47

x

Products for your research

Flow Cytometry Multicolor Selector

With over 2,500 antibodies and kits for flow cytometry and more than 20 fluorochromes...

49

...designing your multicolor experiment has never been easier!http://www.abcam.com/flow-cytometry

Website

Simplify Your Flow Reagent Search

Features

• Over 2,500 conjugated antibodies

• More than 20 fluorophores,

including 6 tandem dyes

• CD and cell-surface targets

• Search up to three targets or clone

numbers at once

50

numbers at once

• Easy filtering to narrow your

selection

• Step-by-step video tutorial

Website

http://www.abcam.com/flow-cytometry

http://www.abcam.com/blog/

Your Complete Resource for Flow

Steptavidin and avidin protein conjugates for detection and purification

Apoptosis and cell viability kits including Annexin V assays, kinetic apoptosis kits, and DNA fragmentation kits

EasyLink conjugation kits for direct staining flow protocols where a conjugated primary for the target of interest is not available, with 25 conjugations available.

51

New range of conjugated secondaries including AlexaFluor

Extensive Technical Resources

• Detailed flow cytometry protocols

• Posters and pathways to aid your research

• Past flow cytometry webinars

Website

• http://www.abcam.com/flow-cytometry

Purification and Detection proteins

Our detection proteins are specific to your target and conjugated to a wide range of enzymes or fluorophores

Avidins • Highly soluble

• Neutral pI and low non-specific binding

• Cy2®, Cy3®, Cy5®, Cy5.5®, Cy7®

• FITC

• His-tag

• HRP

•

52

Streptavidins • PE

• PE – Texas Red

• Spectral Red

• Surelight Allophycocyanin

• TRITC

• 5nm Gold, 10 nm Gold, 15 nm Gold, 20 nm Gold

Deglycosylated

avidins

• Neutral pI and very high affinity for biotin, low unspecific binding

• Alkaline phosphatase

• Allophycocyanin

• Beta – galactosidase

• Cy2®, Cy3®, Cy5®

• FITC

• HRP

• Phycoerythrin

• Rhodamine

• Texas Red

Apoptosis & Cell viability kits for Flow

Quick results

Reproducible data

Apoptosis markers Product?

Phosphatidylserine (PS) exposure

Annexin V-FITC Apoptosis Detection Kit (ab14085)

Kinetic Apoptosis Kit (Flow Cytometry) (ab129816)

53

Kinetic Apoptosis Kit (Flow Cytometry) (ab129816)

Loss of mitochondrial membrane potential (∆Ψm)

JC-10 Mitochondrial Membrane Potential Assay Kit (Flow Cytometry) (ab122133)

Increase of sub-G1 population

Propidium iodide (ab14083)

Cell Cycle Assay Kit (Fluorometric – Green) (ab112116)

Caspase activation Caspase 9 (active) FITC Staining Kit (ab65615)

DNA fragmentation In situ Direct DNA fragmentation assay kit (ab66108)

Find out more:

http://www.abcam.com/apoptosiskits

Fluorescent dyes

Dyes available and flow cytometry features

DRAQ5™ DRAQ7™ CyTRAK Orange™

Labelling of LIVE or fixed cells Labelling of DEAD and permeabilized cells

Labelling of LIVE or fixed cells

Staining of nuclear dsDNA (not mtDNA)

Staining of nuclear dsDNA in dead cells

Differential nuclear and cytoplasmic staining

No compensation needed with No compensation needed with Compensation required

54

No compensation needed with common FITC (or GFP) & PE combinations

No compensation needed with common FITC (or GFP) & PE combinations

Compensation required

Compatible with FITC/PE stained samples

Compatible with FITC/PE stained samples

Blue laser co-excitation with FITC/GFP

Nuclei / Cell Cycle / Ploidy parameter in blood / leukaemia cell subset analysis

Dead cell exclusion – can act as a viability gate

Gate nucleated cells from debris / detection of rare events (two dyes in one)

Find out more:

http://www.abcam.com/DRAQ5

EasyLink Antibody Conjugation Kits

The easiest way to conjugate your antibodies

• A choice of 18 fluorescent labels

• Rapid labelling protocol

• Available in convenient sizes

• 3x10µg

55

• 3x10µg

• 1x100µg

• 3x100µg

• 1x1mg

Find out more:

http://www.abcam.com/EasyLink

Brand new: Alexa Fluor® conjugated secondary antibody range

Alexa Fluor® 488Alexa Fluor® 555

Alexa Fluor® 647Alexa Fluor® 594

ab150157 ab150113ab150151

56

Alexa Fluor® is a registered trademark of Life Technologies.Alexa Fluor® dye conjugates contain(s) technology licensed toAbcam by Life Technologies.

Find out more at www.abcam.com/alexa

Why choose an Abcam Alexa Fluor®

conjugated secondary?

Extensively tested in the Abcam laboratories – to guarantee bright staining and low background

Large selection of pre-adsorbed antibodies-ensuring low species cross-reactivity

Dilution range of 1/200 – 1/1000 (10-2 µg/ml) in

57

Dilution range of 1/200 – 1/1000 (10-2 µg/ml) in IF/ICC – perform at least 250 stainings*

Antibodies conjugated to Alexa Fluor®

488/555/594/647 – for your multi-color imaging experiments

Competitively priced

Find out more at www.abcam.com/alexa

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dyeconjugates contain(s) technology licensed to Abcam by Life Technologies.

How to contact us

US

• Email: [email protected]

• Tel: +1 888-77-ABCAM (22226)or 617-225-2272

• Website: www.abcam.com

Hong Kong


• Tel: +(852) 2603 6823

• Website: www.abcam.cn

Abcam’s Scientific Support Team

58


UK


• Tel: +44 (0) 1223 696000


Japan


• Tel: +81 (0) 3 6231 0940

• Website: www.abcam.co.jp

Allergy and Asthma 2013

Date: May 23-24, 2013

Venue: Bruges, Belgium

Conference Topics

• Innate immune cells in asthma

• Epithelial biology and asthma

• Understanding adaptive immunity in

Oral abstract deadline:February 22, 2013

Poster abstract deadline:March 25, 2013

59

• Understanding adaptive immunity in

asthma

• Environment and asthma

Confirmed speakers

• David Artis (University of Pennsylvania)

• John Fahy (University of California)

• Darryl Knight (University of British

Columbia)

• Carla Ribeiro (University of North Carolina)

and may more....

Meeting website:www.abcam.com/AA2013

Inflammasomes in Health and Disease

Date: June 24-25, 2013

Venue: Boston, US

Conference Topics

• General mechanisms of activation

• Inflammasomes in infection

Oral abstract deadline:April 26, 2013

Poster abstract deadline:May 17, 2013

60

• Inflammasomes in infection

• Inflammasomes and sterile inflammation

• Metabolism-inflammasome link

Keynote speaker:

• Vishva Dixit (Genentech, US)

Meeting chairs:

• Kate Fitzgerald, University of Massachusetts

• Gabriel Nunez, University of Michigan

Meeting website:www.abcam.com/inflammasomes2013

Upcoming webinars

Fluorescent Western Blotting: Introduction and Application

February 28, 2013

15:00 GMT, 10:00 EDT, 07:00 PDT

www.abcam.com/fluorescentWB

61

IHC/ICC Staining Techniques Using Single and Multiple Labels

March 06, 2013

15:00 GMT, 10:00 EDT, 07:00 PDT

www.abcam.com/IHCICCwebinar

Application www.abcam.com/fluorescentWB

Questions?

62

Documents

January 31, 2013 Ian Dimmick (Newcastle University) Graham ...docs.abcam.com/pdf/events/compensation-webinar-slides.pdfCD27 APC CD27 APC KLRg1-A488 KLRg1-A488 CD45ra PB CD27 APC KLRg1-A488