Experimental Design and Considerations for Spectral Flow ...

Josh Croteau, PhDTechnical Applications Scientist IIGWU Cancer Center Flow Core09/18/2019

Experimental Design and

Considerations for Spectral

Flow Cytometry

BioLegend• TotalSeqTM – Novel product line for

antibody mediated protein analysis in scRNAseq applications like CITE-seq.

• SparkTM & FireTM – New fluors to expand the tool chest for advanced multicolor flow cytometry, and, spectral flow cytometry applications.

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Outline• Spectral vs conventional flow cytometry.

• Factors influencing flow panel performance.

• Useful tools for panel design.

• Important flow cytometry controls.

• Using the “tiered” panel design approach.

Representative 7 Color Data:CD3, 4, 8, 19, 56, IgM, CCR6

Courtesy of: Biolegend

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Understanding Spectral vs Conventional Flow Cytometry

Conventional Flow Cytometry• Fluors are excited at specific wavelengths and light collected at specific

“bands” of light (PMTs or APDs).

• Multiplexing is achieved by spatial isolation of detectors, optical configuration, and, compensation.

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Spectral Flow Cytometry• Fluors are excited at specific wavelengths, and, light is collected across

the full spectrum by dedicated detector modules (APD Arrays).

• Multiplexing is achieved by deconvolution of fluorescent spectral “fingerprints” across an array of detectors detecting the full spectrum.

CONFIDENTIAL Courtesy of: Cytek Biosci.

CONFIDENTIAL

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14Blue 488nm laser

3L Aurora Configuration

V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16Violet 405nm laser

R1 R2 R3 R4 R5 R6 R7 R8Red 633nm laser

3L Cytek Aurora• Spatially separated lasers

• Full Spectrum analysis

• 38 Fluor detectors

• 20+ Parameters

CONFIDENTIAL

V1 428/15 Brilliant Violet™ 421

V2 443/15 SuperBright™ 436

V3 456/15 Pacific Blue



V6 528/21

V7 549/22


V9 594/23



V12 692/28




V16 812/34

B1 508/20 BB515

B2 528/21 Alexa Fluor® 488 or FITC

B3 549/22 Spark Blue 550™

B4 571/23 PE

B5 594/23

B6 618/24 PE/Dazzle™ 594

B7 660/17 PE/Cy5

B8 678/18 PerCP

B9 697/19 PerCP/Cy5.5 or TBD

B10 717/20 PerCP/eF™ 710 or TBD

B11 738/21

B12 760/23

B13 783/23 PE/Cy7

B14 812/34 TBD

R1 660/17 APC

R2 678/18 Alexa Fluor® 647

R3 697/19 Spark NIR 685™


R5 738/21

R6 760/23 Zombie NIR

R7 783/23 APC/Fire™ 750

R8 812/34 TBD

5L Cytek Aurora

CONFIDENTIAL

UV1 372/15 V1 428/15 Brilliant Violet™ 421

UV2 387/15 BUV395 V2 443/15 SuperBright™ 436

UV3 427/15 V3 458/15 Pacific Blue™



UV6 473/15 BUV496 V6 525/17

UV7 514/28 V7 542/17


UV9 581/31 BUV563 V9 598/20


UV11 664/27 BUV661 V11 664/27 Brilliant Violet™ 650

UV12 691/28 V12 692/28


UV14 750/30 BUV737 V14 750/30 Brilliant Violet™ 750


UV16 812/34 BUV805 V16 812/34

B1 508/20 BB515 YG1 577/20 PE

B2 525/17Alexa Fluor® 488

or FITC YG2 598/20

B3 542/17 Spark Blue 550™ YG3 615/20 PE-Dazzle™ 594

B4 581/19 PE YG4 660/17 PE/Cy5

B5 598/20 YG5 678/18 TBD

B6 615/20 PE/Dazzle™ 594 YG6 697/19

B7 660/17 PE/Cy5 YG7 720/29

B8 678/18 PerCP YG8 750/30

B9 697/19PerCP/Cy5.5 or

TBD YG9 780/30 PE/Cy7

B10 717/20PerCP/eF™ 710 or

TBD YG10 812/34

B11 738/21

B12 760/23

B13 783/23 PE/Cy7 R1 660/17 APC

B14 812/34 TBD R2 678/18 Alexa Fluor® 647

R3 697/19 Spark NIR 685™


R5 738/21

R6 760/23 Zombie NIR

R7 783/23 APC/Fire™ 750

R8 812/34 TBD

Spectral “Signatures”• Emission for each dye is

measured off of every laser.

• The data are stitched together to show the entire “signature”.

• These signatures are used to unmix.

CONFIDENTIAL

APC

ZombieTM NIR

Courtesy of: Cytek Biosci.

Violet Blue Red

Linear Unmixing

CONFIDENTIAL

• Aurora uses linear unmixing – what is this?

– Mathematical technique that decomposes multiple elements in an unknown by analyzing their makeup relative to a library of reference standards (single color controls).

• In spectral cytometry, this is used to decompose the fluorescent components in a multicolor sample using spectral reference controls as the standards.

From “Linear Unmixing for Dummies”https://microscopy.jhmi.edu/Learn/refman/Zeiss/LSM510Meta/Linear_Unmixing_for_Dummies2.pdf

https://microscopy.jhmi.edu/Learn/refman/Zeiss/LSM510Meta/Linear_Unmixing_for_Dummies2.pdf

Compensation vs Spectral Unmixing

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• Single color controls used to calculate % contribution of light from non-primary fluor into primary detector.

• For N color assay, N detectors are needed.

• A compensation matrix is calculated: it is a square matrix, N times N.

FITC PE

FITC into PE spillover

• Single color controls to establish reference standards.

• Unmixing determines which combination of these controls best fits the multicolor spectral signature of the full stained sample.

• The number of detectors has to be higher than the number of fluorochromes.

• An unmixing matrix is calculated: in the case of Aurora, it is an N times 38 matrix

FITC

PE


Factors Influencing the Performance and Utility of Multicolor Flow Cytometry

&

Useful Tools for Panel Design

Fluorescent Molecules Categories

Synthetic Organic dyes:Alexa Fluor®, Cyanine, FITC, DyLight, CF® Dyes

Fluorescent Proteins: PE, APC, PerCP,

& Tandems

Organic Polymer dyes: Brilliant Violet®,

Brilliant UV®, Including Tandems

FITC Alexa Fluor® 594 Allophycocyanin (APC)

~0.3 – 2 kDa ~ 60-80 kDa ~ 35 – 250 kDa

CONFIDENTIAL

Synthetic Organic dyes• Advantages:

– Small

– Resilient to formalin and alcohol fixatives

– Highly photostable

• Disadvantages:

– Limited brightness for many formats

• Higher F:P (3-6+)

CONFIDENTIAL

FITC

Alexa Fluor® 594

FITC


Organic Polymer Dyes• Advantages:

– Bright (chain of fluors)

– Tunable using tandem chemistry

– Base polymers fixative resilient and stable

• Disadvantages:

– Expense

– Families contain tandems

– Larger

• Variable F:P


Fluorescent Proteins• Advantages:

– Bright (PE/APC)

– Tunable using tandem chemistry

– Affordable

• Disadvantages:

– Alcohol denatures

– Families contain tandems

– Large

• Low F:P (1-2)

CONFIDENTIAL

Allophycocyanin (APC)


Cytek Fluor Selection Guide• These signatures are all listed in Cytek’s Fluor Selection Guide

• They help you to inform your panel design

• These are directly off the cytometers – showing just the MFI in each detector

• You can find the fluor guides here: www.cytekbio.com/blogs/resources


BV421

http://www.cytekbio.com/blogs/resources

Spectral Consideration: Tandems• Tandem dyes utilize FRET (Forster resonance

energy transfer) to pass the excitation energy at a specific wavelength from one fluor(donor) to another (acceptor).

• Of special consideration in spectral flow because the donor fluor is often shared among many fluors:

– PE, PE/Dazzle594, PE/Cy5, PE/Cy5.5, PE/Cy7

– PerCP, PerCP/Cy5.5

– BV421, BV605, BV650, BV711, BV785

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Spectral Consideration: Tandems

CONFIDENTIAL

APC

Alexa Fluor 647


PerCP

PerCP-Cy5.5


BioLegend Aurora Spectral Viewer• % Normalized Excitation/Emission.

• Good alternative way to evaluate spectral similarities/differences.

• www.biolegend.com/en-us/aurora-spectra-analyzer

CONFIDENTIAL

APC/FireTM 750 vs ZombieTM NIR

http://www.biolegend.com/en-us/aurora-spectra-analyzer

Spectral Consideration: Tandems• APC Excitation off of the

405nm laser is unique compared to AF647.

• PerCP and PerCP/Cy5.5 have similar characteristics, close peak emission, and, dissimilar brightness.

CONFIDENTIAL

APC vs Alexa Fluor 647

PerCP vs PerCP/Cy5.5

Spreading Error• Errors in photon counting arise when using highly overlapping dyes/channels.

Specific to dye, not antigen.

• Compensation/unmixing reveals spread, does not create it!

• Dim antigens impacted more heavily.

• Understanding spread is critical to designing multi parameter experiments.

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Spreading ErrorMulticolor Scenario, spread into Fluor C from Fluor A or B:

• Left: spread from Fluor A into Detector C.

• Right: spread from Fluor B into Detector C.

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Antigen X Fluor A

An

tige

nY

Flu

or

C

Antigen X Fluor B

An

tige

n Y

Flu

or

C

Count

An

tige

n Y

Flu

or

C

Count

An

tige

n Y

Flu

or

C

No Reduction in SI Due to SPREAD from Antigen X, Fluor A

Reduction in SI Due to SPREAD from Antigen X, Fluor B

SI SI SI SI

Spreading Error – Incorporating the Full Spectrum

• PE-Cy5 spill (spread) into APC.

CONFIDENTIAL

PE-Cy5

APC

Raw Signatures

Normalized Signatures

Fluorochrome/ Spillover

V11 V12 V13 B7 B8 B9 R1 R2 R3

PE-Cy5 28% 17% 10% 74% 100% 74% 38% 61% 53%

APC 14% 4% 4% 1% 1% 1% 100% 80% 60%

Unmixed data

Count

AP

C SI

Spreading Matrix – Cytek 3L Matrix (24 Color)

CONFIDENTIAL

• The fluor in the row impacts the one in the column.

• Red means the fluor in that row has significant spread into the dye in the column.


Spreading Matrix – BioLegend 3L Matrix (23 Color)

CONFIDENTIAL

• Generated using anti-CD4 in every fluor.

• More red = more significant reduction in staining index with that combination.

Marker Antigen Density > CD4

Better Able to “survive” Spread

Marker Antigen Density < CD4

Less Able to “survive” Spread

Spreading Matrix – BioLegend 3L Matrix (23 Color)

CONFIDENTIAL

• Generated using anti-CD4 in every fluor.

• More red = more significant reduction in staining index with that combination.

PE into APC Stain index reduction= 0%

PE

APC

PE into BV570 Stain index reduction= 83%

PE

BV570


Important Controls For Multicolor Spectral Flow Cytometry

Controls

CONFIDENTIAL

• Your experiment is only as good as your controls.

– Instrumentation Controls: Single Stained, Negative/Unstained

– Specificity & Gating Controls: Unstained, Isotype, Isoclonic, FMO

– Biological Controls: Negative and Positive controls, time course.

• MUST HAVE FOR THE AURORA:

– Unstained cellular control. Needed for autofluorescence subtraction. Consider unstained positive biological controls (activation, treatment??).

– Single stained controls. Can be beads or cells, cells can be better for some.

Single Color Controls – Spectral Unmixing

CONFIDENTIAL

• Positive and negative particles must be clearly separated.

• Positive and negative particles have IDENTICAL autofluorescencecharacteristics.

• Sufficient events for both data points (5000).

• Spectrum of positive control (single stain) must be IDENTICAL to the spectrum for the same fluor in the multicolor sample.

• Do NOT substitute fluors. Match lots whenever possible; especially for tandems.

Single Color Controls – Beads vs Cells

CONFIDENTIAL

Cells unmixed with beads

Cells unmixed with cells

Spark

685-A

Spark

685-A

• Beads can often be used as single color controls for spectral unmixing.

Single Color Controls – Beads vs Cells

CONFIDENTIAL

• There are times where cells are required.

• Fluor and antigen/target dependent.

Cells unmixed with beads

Cells unmixed with cells

Sp

ark

68

5-A

Sp

ark

68

5-A

Single Color Controls – Accurate Unmixing

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• Be sure your positive and negative gates contain the correct population.

Single Color Controls – Accurate Unmixing

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• Changing Gain can alter the spectral signature. Only do so across all detectors.

Managing Spill: Single Color Controls

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• Single stained controls tell you the behavior of the antigen on a fluor and the impact of that pairing (antigen on fluor) on other channels (spread).

Reagent and Gating Controls: Isotype

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• Indicator of non-specific binding due to charge interactions of the antibody and/or fluor, and, possibly Fc binding.

• Does not demonstrate antibody specificity/background. Antibody is a different clone raised against a different antigen and has different structure.

Be weary of Isotypes:In this example the

Isotype has stronger “staining” than background and

specific antibody.

Andersen et al. (2016) Cytometry A 89:1001-1009.

https://www.ncbi.nlm.nih.gov/pubmed/27731950

Reagent and Gating Controls: Isoclonic

CONFIDENTIAL

• Set of several controls using the same antibody clone. Fluor conjugated, and, unconjugated are mixed in competing ratios in order to out-compete fluorescently labeled antibody. Demonstrates antibody specificity and true non-specific/Fc background.

Anti-Hu CD20 IsoclonicExperiment:

Black – FITC Alone Green – FITC:Pure, 5:1

Blue – FITC:Pure, 1:1Red – FITC:Pure, 1:5

Courtesy of: Jessica Gucwa, JHMI

Reagent and Gating Controls: FMO

CONFIDENTIAL

• Used to establish positive and negative staining in the context of a fully stained panel, accounts for spectral overlap from all sources after compensation/unmixing (autofluorescence, spreading error, ect).

Courtesy of: Mario Roederer, NIH

Accounts for Spread from all other channels into

your primary channel of interest.

Biological Controls

CONFIDENTIAL

• Needed for “variable” markers that are tightly regulated. Expression may be transient (temporally), or, very modest. Need true positives and negatives.

• Experiment specific. Consider at least incorporating a true biological negative and positive. Can sometimes be cells from the same sample

• In Spectral, especially important for Autofluorescence subtraction.

https://www.nature.com/articles/s41598-018-30623-2/figures/5

https://www.nature.com/articles/s41598-018-30623-2/figures/5

Biological Controls

CONFIDENTIAL

• Veri-Cells PBMC activated with mitogen cocktail in the presence of monensin

Activated ActivatedIsotype Isotype

Titration – ZombieTM Example

CONFIDENTIAL

Negative Cells

• Without titration we have Zombie positive “live” cells.

• How will this impact unmixing??


Pulling It Together: Tiered Approach to Flow Panel Design

Spread

CONFIDENTIAL

• The overall goal of strategic panel design is to manage spread in order to maximize resolution in each parameter.

• Spread is worsened by:

– Inappropriate single color controls for comp/unmixing.

– Poor antigen and fluor pairings.

– Improperly balanced detectors.

– Lack of reagent titration.

– Excess parameters surpassing feasibility.

– Intracellular or phospho staining.

Bad Combo:CD8 BV510 CD27 APCCD4 Spark 685 CD127 AF647

Validated 22 Color Combination

CONFIDENTIAL

BioLegend Validated Fluors – 3L AuroraViolet 405 nm Blue 488 nm Red 633 nm

Brilliant Violet™ 421 FITC or Alexa Fluor® 488 APCPacific Blue™ Spark Blue™ 550 Alexa Fluor® 647Brilliant Violet™ 510 PE Spark NIR™ 685Brilliant Violet™ 570 PE/Dazzle 594 Alexa Fluor® 700Brilliant Violet™ 605 PE/Cyanine5 APC/Fire™ 750Brilliant Violet™ 650 PerCP/Cyanine5.5 Zombie™ NIRBrilliant Violet™ 711 PE/Cyanine7Brilliant Violet™ 750Brilliant Violet™ 785

Panel Design

CONFIDENTIAL

• Conventional Design techniques still apply!

1. Start with the BIOLOGY!

– Antigen Classification: Primary, Secondary and Tertiary (Expression Level).

– Draw out gating strategy for cells and markers of interest.

– Predict antigen co-expression.

2. Assign Fluors

– How many antigens do we need to detect?

– Which highly overlapping fluors can I avoid?

3. What antibodies are commercially available for each antigen ?

– Use Panel Selector or Panel Builder to mine BL catalog.

– Adjust according to availability.

Panel Design

CONFIDENTIAL

1. Start with the BIOLOGY!

Classify Targets based on level of expression

- Primary Ag = High expression, “Bright”

- Secondary Ag = Moderate/variable/unknown expression

- Tertiary Ag = Low expression or few options for fluors (rare), “Dim”

Determine gating strategy

- What cell types am I interested in? What are their phenotypes?

- Which markers will be co-expressed? IE where do we avoid excess spread?

Web Resources

CONFIDENTIAL

Biolegend Essential Markers Webpage:

Web Resources

CONFIDENTIAL

Biolegend Cell Markers:

Fluor Brightness – Cytek 3L


Working Example: Biolegend & Cytek

CONFIDENTIAL

Human DC panel

Modified from OMIP-044; 28-color Immunophenotyping of Human DC compartment – Mair & Prlic, 2018

• Gate on canonical DC subpopulations

• Lineage markers to enumerate monocytes, B cells, NK cells, CD4 & CD8 T cells

• Assess changes in DC phenotype based on activation

Courtesy of: Leesa Penell & Cytek Biosci.


CONFIDENTIAL

22 Parameters In Total:

• Basic: CD45, Live/Dead

• Lineage: CD3, CD19, CD56

• T cells: CD4, CD8, CD45RA, CCR7

• Myeloid/DC markers: CD14, CD11b, CD16, CD123, CD11c, HLA-DR, CD141, CD1c

• DC phenotyping: CD40, CD80, CD86, Sirp-α (CD172)



CONFIDENTIAL

Predicted Gating Strategy


CD

45

Live/dead

CD

14

CD16

CD

3

CD19

CD

45

RA

CCR7

HLA

-DR

CD80

CD

56

CD123

HLA

-DR

CD11c

CD

14

1

CD1c

CD45+ Live CD14-

CD19+

CD

4

CD8

CD3+ CD4 or CD8 +

CD14- CD3- CD19-

naïve

pDC

CD141 cDC

CD1c cDC

NK

Lin- CD11c+HLA-DR+Histograms on DC subsets for:- CD40- CD80- CD86- Sirp-α

DN DCs

CD14+

CONFIDENTIAL Courtesy of: Leesa Penell & Cytek Biosci.

Violet Excited Fluors Blue Excited Fluors Y/G Excited Fluors Red Excited Fluors

BV421 – CD1c BB515 – CD40 PE – Sirp-α (CD172) APC – CD141

SB436 – CD123 AF488 – CD45RA PE/Dazzle 594 – CD11c AF647 – CD86

eFluor450 – CD16 AF532 – CD3 PE/Cy5 – CD80 AF700 – CD45

BV480 PerCP-Cy5.5 PE/Cy7 – CCR7 APC/Fire750 – HLA-DR

BV510 PerCP-eFluor710 – CD11b Zombie NIR

BV570 – CD8

BV605 – CD56

BV650 – CD19

BV711 – CD14

BV750 – CD4

BV785

Working Example: First Design




SB436 – CD123 AF488 – CD45RA PE/Dazzle 594 – CD11c AF647 – CD86




BV570 – CD8

BV605 – CD56

BV650 – CD19

BV711 – CD14

BV750 – CD4

BV785

Working Example: Troublesome Pairings




SB436 – CD123 AF488 – CD45RA PE/Dazzle 594 – CD11c AF647




BV570 – CD8

BV605 – CD14

BV650 – CD19

BV711 – CD56

BV750 – CD4

BV785 – CD40

Working Example: Revised Design - Testing



BV421 – CD1c BB515 – CD11c PE – Sirp-α (CD172) APC – CD141

SB436 AF488 – CD45RA PE/Dazzle 594 – CD123 AF647



Zombie Aqua PerCP-eFluor710 – CD11b

BV570 – CD8

BV605 – CD14

BV650 – CD19

BV711 – CD56

BV750 – CD4

BV785 – CD40

Working Example: Final Design after testing


CONFIDENTIAL

Predicted Gating Strategy


CD

45

Live/dead

CD

14

CD16

CD

3

CD19

CD

45

RA

CCR7

HLA

-DR

CD80

CD

56

CD123

HLA

-DR

CD11c

CD

14

1

CD1c

CD45+ Live CD14-

CD19+

CD

4

CD8

CD3+ CD4 or CD8 +

CD14- CD3- CD19-

naïve

pDC

CD141 cDC

CD1c cDC

NK

Lin- CD11c+HLA-DR+Histograms on DC subsets for:- CD40- CD80- CD86- Sirp-α

DN DCs

CD14+


CD80 CD40Sirp-α

Representative DataNot Shown:T cell MemoryB cells

Panel Design - Review

CONFIDENTIAL

• Conventional Design techniques still apply!

• Start with theoretical panel.

• Compare the controls – single stains to the multicolor to assess spread.

• Re-design as needed to eliminate challenging combinations and maximize resolution.

• Perform full panel stain and analyze data.

• Stain smaller ‘sub-panels’ as needed

• Don’t be scared to revise!

Closing Thoughts• Tough Combinations on the 3L Aurora:

BV570

PE-Cy5AF 647

BV421

BB515 AF488

SB436

PE

CONFIDENTIAL

• PE & BV570

• PE/Cy5 & AF647

• BB515 & AF488

• BV421 & SB436

Closing Thoughts

CONFIDENTIAL

1. Before applying unmixing, check for anything obviously wrong with the spectrum. You can use a printed reference control for your machine with your gain settings.

2. Don’t change your gain settings unless its changed the same % across every detector in the array

3. Run both beads and cells as single color controls.

4. Prior to full analysis mode, check ALL single color controls, including unstained cells.

5. Gating controls (FMOs) still important, but not minus one anymore. Consider subtracting multiple fluors (sub-panels, or FM-Multiples).

Closing Thoughts

CONFIDENTIAL

• Controls are extremely important

– Ensure your single stained samples have enough positive and negative events.

– It’s ok to use a mix of beads and cells for your controls, just be sure to use the appropriate negative!

• Overlap will still be a problem – choose fluors carefully!

– Can’t just throw every dye in and expect that unmixing fixes everything.

Disclaimer

CONFIDENTIAL

What a good panel can do for you

Thank you!Questions & Comments?

Josh Croteau, Ph.D.Technical Applications Scientist II

BioLegend Inc.Mobile: 858-361-4133

Email: [email protected]

Custom Conjugations

CONFIDENTIAL

Yield/Percent Recovered After

Purification

Cost Per Milligram Purified

Antibody/Protein

Synthetic Organic Dyes

(FITC, Alexa®, ect)

Fluorescent Proteins and Tandems (PE, APC, ect)

Organic Polymer Dyes

(BV®, ect)

$$$$

$

+

++++

Experimental Design and Considerations for Spectral Flow ...

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