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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.
CONFIDENTIAL
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
CONFIDENTIAL
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.
CONFIDENTIAL
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
V4 473/15 Brilliant Violet™ 480
V5 508/20 Brilliant Violet™ 510
V6 528/21
V7 549/22
V8 571/23 Brilliant Violet™ 570
V9 594/23
V10 618/24 Brilliant Violet™ 605
V11 664/27 Brilliant Violet™ 650
V12 692/28
V13 720/29 Brilliant Violet™ 711
V14 750/30 Brilliant Violet™ 750
V15 780/30 Brilliant Violet™ 785
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™
R4 717/20 Alexa Fluor® 700
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™
UV4 443/15 V4 473/15 Brilliant Violet™ 480
UV5 458/15 V5 508/20 Brilliant Violet™ 510
UV6 473/15 BUV496 V6 525/17
UV7 514/28 V7 542/17
UV8 542/28 V8 581/19 Brilliant Violet™ 570
UV9 581/31 BUV563 V9 598/20
UV10 612/31 V10 615/20 Brilliant Violet™ 605
UV11 664/27 BUV661 V11 664/27 Brilliant Violet™ 650
UV12 691/28 V12 692/28
UV13 720/29 V13 720/29 Brilliant Violet™ 711
UV14 750/30 BUV737 V14 750/30 Brilliant Violet™ 750
UV15 780/30 V15 780/30 Brilliant Violet™ 785
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™
R4 717/20 Alexa Fluor® 700
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
Compensation vs Spectral Unmixing
CONFIDENTIAL
• 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
Courtesy of: Cytek Biosci.
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
Courtesy of: Cytek Biosci.
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
CONFIDENTIAL Courtesy of: Cytek Biosci.
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)
Courtesy of: Cytek Biosci.
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
CONFIDENTIAL Courtesy of: Cytek Biosci.
BV421
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
CONFIDENTIAL
Spectral Consideration: Tandems
CONFIDENTIAL
APC
Alexa Fluor 647
Courtesy of: Cytek Biosci.
PerCP
PerCP-Cy5.5
Courtesy of: Cytek Biosci.
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
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.
CONFIDENTIAL
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.
CONFIDENTIAL
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.
Courtesy of: Cytek Biosci.
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
Courtesy of: Cytek Biosci.
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
CONFIDENTIAL
• Be sure your positive and negative gates contain the correct population.
Single Color Controls – Accurate Unmixing
CONFIDENTIAL
• Changing Gain can alter the spectral signature. Only do so across all detectors.
Managing Spill: Single Color Controls
CONFIDENTIAL
• 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
CONFIDENTIAL
• 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.
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
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??
Courtesy of: Cytek Biosci.
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
CONFIDENTIAL Courtesy of: Cytek Biosci.
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.
Working Example: Biolegend & Cytek
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)
Courtesy of: Leesa Penell & Cytek Biosci.
Working Example: Biolegend & Cytek
CONFIDENTIAL
Predicted Gating Strategy
Courtesy of: Leesa Penell & Cytek Biosci.
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
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: Troublesome Pairings
CONFIDENTIAL Courtesy of: Leesa Penell & Cytek Biosci.
Violet Excited Fluors Blue Excited Fluors Y/G Excited Fluors Red Excited Fluors
BV421 – CD1c BB515 – CD86 PE – Sirp-α (CD172) APC – CD141
SB436 – CD123 AF488 – CD45RA PE/Dazzle 594 – CD11c AF647
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 – CD14
BV650 – CD19
BV711 – CD56
BV750 – CD4
BV785 – CD40
Working Example: Revised Design - Testing
CONFIDENTIAL Courtesy of: Leesa Penell & Cytek Biosci.
Violet Excited Fluors Blue Excited Fluors Y/G Excited Fluors Red Excited Fluors
BV421 – CD1c BB515 – CD86 PE – Sirp-α (CD172) APC – CD141
SB436 – CD123 AF488 – CD45RA PE/Dazzle 594 – CD11c AF647
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 – CD14
BV650 – CD19
BV711 – CD56
BV750 – CD4
BV785 – CD40
Working Example: Revised Design - Testing
CONFIDENTIAL Courtesy of: Leesa Penell & Cytek Biosci.
Violet Excited Fluors Blue Excited Fluors Y/G Excited Fluors Red Excited Fluors
BV421 – CD1c BB515 – CD11c PE – Sirp-α (CD172) APC – CD141
SB436 AF488 – CD45RA PE/Dazzle 594 – CD123 AF647
eFluor450 – CD16 AF532 – CD3 PE/Cy5 – CD80 AF700 – CD45
BV480 PerCP-Cy5.5 PE/Cy7 – CCR7 APC/Fire750 – HLA-DR
Zombie Aqua PerCP-eFluor710 – CD11b
BV570 – CD8
BV605 – CD14
BV650 – CD19
BV711 – CD56
BV750 – CD4
BV785 – CD40
Working Example: Final Design after testing
Working Example: Biolegend & Cytek
CONFIDENTIAL
Predicted Gating Strategy
Courtesy of: Leesa Penell & Cytek Biosci.
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.
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)
$$$$
$
+
++++