Introduction to Spectral Cytometry using Cytek® Aurora and Northern Lights

Day 3

What happens when I have too much caffeine!

A B cell panel for human PBMCs

Marker FluorLevel of Expression /

Cell Frequency

Control: Beads

or CellsControl Stained With

CD19 BV510 High/ intermediate Cells Any bright BV510IgD BV421 High/ intermediate Cells Any bright BV421

CD27 BV785 Intermediate/ high Cells CD27 BV785, same lotCD62L BV605 High/ high Cells CD62L BV605, same lotCD38 Pacific Blue High/intermediate Cells Any bright Pacific BlueIgM BV650 High/ intermediate Cells IgM BV650, same lotIgG B711 Low/ low Beads IgG BV711, same lot

unstained PBMCs

Intracellular Cytokine Staining/Proliferation Assay, cultured PBMCs

Marker Fluor Level of Expression /

Cell Frequency

Control: Beads

or Cells

Control Stained With

CFSE High/high Unstim cells CFSE well titrated*CD3 PE CF594 Low/ high Unstim cells CD3 PE-CF594, same lotCD4 APC Low/high Unstim cells Any bright APC, but CD4 is goodCD8 APC R700 Intermediate/high Cells CD8 APC-R700, same lotIFNg efluor 450 Intermediate/ int. Stim cells Any bright eFluor 450IL-4 PE Cy7 High/low Stim cells IL-4 PE-Cy7, same lot, higher stopping gate for enough events

IL-17 PerCP e710 Low/low Beads IL-17 PerCPeFluor710, same lotviability Aqua Amine Dye high/low Cells Aqua Amine Dye

unstained PBMCs cultured Unstim and stimulated

Separate controls

*Switch CFSE to Cell Trace Violet – much easier to titrate into range

Cells Beads for Surface Ags Beads for i.c. Ags

1. Add surface Abs

2. Incubate/wash

3. Fix Perm

4. Wash

5. AF647 Ab

6. Incubate/wash

7. Run!

You are planning to perform an assay for detection of FOXP3 and to follow the protocol below. Please write down

the protocol you will follow (step by step) to stain beads to be used as controls for surface antigens AND for

intracellular antigens

Reference Control QC

Positive peak Spectrum

Negative peak Spectrum

PE control: Good

Reference Control QC

PE control: neg peak has pos signal – ALWAYS WASH YOUR BEADS/CELLS

Reference Control QC

Super Bright 780 Control: not highest peak gated, highest peak has incorrect signature (has SB436 sig) tandem degradation, pos peak in bottom plot is the donor, the acceptor is no longer fluorescing

Reference Control QC

BB700 Control: Pos gate too wide, too heterogeneous, plot 2 and 3 show different spectra, lower peak looks like carry over

Reference Control QC

PE-Cy5 Control: a lot of tailing on pos peak – usually when beads get stuck on the side of the tube and encounter variable amounts of Ab, this is also a problem with old beads that don’t bind uniformly

Knowledge Review

• Analyze full spectrum signatures✓Understand how full spectrum signatures are generated

✓Identify unique signatures

✓Normalize spectrum signatures

✓QC and troubleshoot signatures

• Understand the fundamentals of spectral unmixing✓How it works

✓How it compares to compensation

✓Understand the workflow in Spectroflo

✓Understand the requirements for optimal reference controls

✓How to QC reference controls

Objectives

By the end of Day 3, you should be able to:

• Understand how to plan and design a multicolor panel✓Understand the fundamentals of panel design

✓Understand the tools available for fluorochrome selection and successful panel design

✓Panel performance evaluation and optimization strategies

Know the BiologyPlanning a Multicolor Experiment

Panel Design: Gathering Information

Identify Antigens• Level of expression: primary, secondary, tertiary

• Antigen co-expression

Select Fluorochromes• Unique signature

• Brightness

• Spread considerations

Check Availability • Are the reagents available in the antibody/fluor combination

SecondaryPrimary TertiaryCD3CD4CD8

CD45RA CCR7

CD127HLA-DR

PD-1CD25

TCR g/d

Level of Antigen Expression

Mahnke, Y. and Roederer, M. Clin Lab Med. 2007 September ; 27(3): 469

Antigen Classification

High densityon or off expression

Intermediate densitycontinuous expression

Low densityunknown expression

CD4 CD127 CD25

Flurochrome Brightness

Know your co-expression

Selecting Fluorochromes with Unique Signatures

Several tools available to

help:

1. Fluorochrome selection

guidelines

2. Online spectrum viewer

3. Similarity & complexity

indices

Blue Laser Unique SignaturesLook at and Identify Unique dyes to use

Fluorochrome selectionStain index

Assessing Brightness: Stain Index Ranking of 65 DyesSta

in In

dex

Dim Mid Bright Very Bright

Example: 35 Fluorochrome Selection

Approx. Emission Wavelength (nm)

UV Violet Blue Yellow Green Red

395 BUV395

420 BV421440 Super Bright 436450 Live/Dead Blue eFluor 450

480 BV480500 BUV496 BV510 BB515

520 FITC

550 Pacific Orange CF514570 BUV563 BV570 PE580 CF568600 BV605 PE/Dazzle594

660 BUV661 BV650 APC680 PerCP PE-Cy5 Alexa Fluor 647

690 PerCP-Cy5.5700 BV711 PerCP-eFluor710 APC-R700

730 BUV737750 BV750780 BV785 PE-Cy7 APC-Fire 750

800 BUV805 Qdot 800

Fluorochrome selectionUnderstanding Spread

SINGLE COLOR SCENARIO

Marker A Fluor X

Marker A Fluor Y

MFI Fluor X

Co

un

ts

MFI Fluor Y

Co

un

ts

Main Contributors for Resolution Reduction

• Instrument Performance

• Instrument Setup

• Fluorochrome Brightness

MULTICOLOR SCENARIO

Marker A Fluor XSINGLE STAINED

Marker A Fluor XMULTICOLOR TUBE

MFI Fluor X

Co

un

ts

MFI Fluor X

Co

un

ts

Main Contributors for Resolution Reduction

• Spread

• Antibody titer

Assessing Antigen Resolution

SINGLE COLOR SCENARIO

Marker A Fluor X

Marker A Fluor Y

MFI Fluor X

MFI

Flu

or

Y

MFI Fluor X

MFI

Flu

or

Y

Stain Index Marker A Fluor X

Stain Index Marker A Fluor Y

MULTICOLOR SCENARIO

Fluor X DOES NOT spread into Fluor Y

Fluor Y DOES NOT spread into Fluor X

MFI

Flu

or

Y

Stain Indices Unchanged

MFI Fluor X

Fluor X DOES SPREAD into Fluor Y

Fluor Y DOES NOT spread into Fluor X

MFI

Flu

or

Y Stain Index Marker A Fluor YDECREASES in presence of fluor X

MFI Fluor X

Considerations

• CO EXPRESSION

• ANTIGEN LEVEL OF EXPRESSION

• Data used for calculations has to be unmixed using a certain combination of fluorochromes

Quantification of Impact of Spread in Resolution

Cross Stain Index Calculation

Fluor X DOES

SPREAD into Fluor Y

Fluor Y DOES NOT

spread into Fluor X MFI Flu

or

Y Stain Index Marker A Fluor Y

DECREASES in presence of fluor X

MFI Fluor X

By unmixing CD4 stained cells

with multiple fluors we can

assess how much the positive

population spread into other

dyes (rSD) .

1 2

Spread % Reduction

We use that spread to calculate how much the

single stained Stain Index (1) is reduced by the

spread introduced by another dye (2)

25

Cross Stain Index Matrix: 35 Color Panel

Dye in row impacts dye in column

BU

V3

95

LIV

E D

EAD

Blu

e

BU

V4

96

BU

V5

63

BU

V6

61

BU

V7

37

BU

V8

05

BV

42

1

Sup

er

Bri

ght

43

6

eFl

uo

r 4

50

BV

48

0

BV

51

0

Pac

ific

Ora

nge

BV

57

0

BV

60

5

BV

65

0

BV

71

1

BV

75

0

BV

78

5

Qd

ot

80

0

BB

51

5

FITC

CF5

14

Pe

rCP

Pe

rCP

-Cy5

.5P

erC

P-e

Flu

or

71

0P

E

CF5

68

PE-

Daz

zle

594

PE-

Cy5

PE-

Cy7

AP

C

Ale

xa F

luo

r 6

47

AP

C-R

70

0

AP

C-e

Flu

or

78

0

BUV395LIVE DEAD Blue

BUV496BUV563BUV661BUV737BUV805BV421

Super Bright 436eFluor 450

BV480BV510

Pacific OrangeBV570BV605BV650BV711BV750BV785

Qdot 800BB515FITC

CF514PerCP

PerCP-Cy5.5

PerCP-eFluor 710

PECF568

PE-Dazzle594PE-Cy5PE-Cy7

APCAlexa Fluor 647

APC-R700APC-eFluor 780

No

Impact

High

Impact

Unmixed CD4 APC

APC

BU

V395

Unmixed CD4 PE-Cy7

BU

V805

PE-Cy7

35-Color Panel

Approx. Emission Wavelength (nm)

UV Violet Blue Yellow Green Red

395 CD45RA BUV395420 PD-1 BV421

440 CD123 SB436

450 Live/Dead Blue CD161 eFluor 450480 IgD BV480500 CD16 BUV496 CD3 BV510 CD86 BB515520 CD57 FITC550 CD20 Pacific Orange CD19 CF514570 CD14 BUV563 HLA DR BV570 CD335 PE

580 CD4 CF568600 CD28 BV605 CD24 PE Dazzle594

660 CD11c BUV661 CXCR3 BV650 CD27 APC

680 CD45 PerCP CD95 PE-Cy5 CD33 Alexa647690 CD11b PerCP Cy5.5700 CCR6 BV711 TCR γδ PerCP eFluor710 CD127 APC-R700

730 CD56 BUV737750 CXCR5 BV750780 CCR7 BV785 CD25 PE Cy7 CD38 APC-eFluor 780

800 CD45RO BUV805 CD8 Qdot 800

Primary Secondary Tertiary

Co-expression and antigen classification are needed for correct fluorochrome choice.

Panel Design and Highly Overlapping Dyes (1)

No SB436+ SB436

NOT co-expressed+ SB436

Co-expressed

TC

Rg

dB

V421-A

No SB436 or eFluor450

+ eFluor450 Co-expressed

+ SB436 NOT co-expressed

+ SB436Co-expressed

Panel Design and Highly Overlapping Dyes (2)

Panel EvaluationMaking sure your panel is optimal

Single Stained Controls allow the full evaluation of:

• Antibody performance/titer/staining conditions

• Adequacy of reference/compensation controls

• Quality of panel design

Marker A Fluor XSINGLE STAINED

Marker A Fluor XMULTICOLOR TUBE

MFI Fluor X

Co

un

ts

MFI Fluor X

Co

un

ts

❖ TAKE THE TIME TO ANALYZE FOR EACH MARKER

❖ THE SINGLE STAINED TUBE vs. THE MULTICOLOR TUBE IS THE FOUNDATION FOR MC PANEL OPTIMIZATION

Single Stained Controls

Evaluation of Single Stained Controls vs Multicolor

32

Multicolor

Single Color CD4 CF568 CD57 FITCCD38 APCeF780 CD45RA BUV395

Evaluation of Single Stained Controls vs Multicolor

33

Multicolor

Single Color CD95 PE-Cy5 CD28 BV605TCRγδ PerCPeF710 CD56 BUV737

Exercise 3

Aims:• Demonstrate your panel design Kung Fu!

• You don’t need to know NK cell biology

• As a general guide anything with a CD# higher than 200 is a tertiary marker, the rest of the Agsare fairly explanatory

Exercise 3 – Panel Design Steps

1. Classify the antigens• Go to vendor websites, check staining examples• Gating strategy

2. Consider co-expression• Gating strategy – know your biology

3. Characterize spread of available fluors – use stain index and spread matrix• Bright fluors that receive low spread• Dim fluors that contribute low spread

4. Assign Fluors• Tertiary – very bright/bright fluors that receive low spread• Secondary

a) bright/mid fluors that receive low spreadb) mid fluors that contribute low spread

• Primary – dim fluors that contribute low spread

Exercise 3 NK Cell Panel Design

Specificity Alt Name PurposeCD2 LFA-2

NK cellsCD16 FCgRIII

CD56 NCAM

CD8

CD3non-NK cells

CD4

CD158a KIR2DL1

NK receptors

CD158b KIR2DL2

CD314 NKG2D

CD335 NKp46

CD337 NKp30

NK Cell Panel

Classify/Fluor Specificity Alt Name PurposeCD2 LFA-2

NK cellsCD16 FCgRIII

CD56 NCAM

CD8

CD3non-NK cells

CD4

CD158a KIR2DL1

NK receptors

CD158b KIR2DL2

CD314 NKG2D

CD335 NKp46

CD337 NKp30

NK Cell Panel

CD3

CD

4 CD3-

CD3+CD4-

CD56

CD

16

CD56C

D8

NK cells

NKT cells

CD2

CD

16

CD158aCD158bCD314CD335CD337

CD

56

CD158aCD158bCD314CD335CD337

CD

56

NK cells

NKT cells

Gating Strategy Exercise 3NK Cell Panel Design

1 2 3 45

Cross Stain Index for 24 Fluorophores

Fluors in the rows spread into fluors in the columns

Fluors in the columns receive spread from fluorsin the rows

Very BrightBrightMidDim

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

* Reagent available

*

*

l Violet 405nm Blue 488 nm Yellow-Green 561 nm Red 640 nmAntigen Fluor Antigen Fluor Antigen Fluor Antigen Fluor

390

430 BV421 (4)

445

460 eF450 (2)

475 BV480 (3)

510 BV510 (2) BB515 (3)

525 FITC (2)

540 AF532 (1)

580 BV570 (2) PE (4)

615 BV605 (2) PE-CF594(4)

660 BV650 (2) APC (4)

680 PE-Cy5 (4)

700 PerCP-Cy5.5 (1)

720 BV711 (3) PerCP-eF710 (3) AF700 (3)

750 BV750 (3)

780 BV785 (3) PE-Cy7 (4) APC-F750 (2)

780 ZombieNIR

810

NK Panel Design Exercise 3PrimarySecondaryTertiary

(1) = Dim(2) = Mid(3) = Bright(4) = Very Bright

Questions?