Optimization of Multicolor Panel

Design Incorporating Low Receptor

Density Antigens

Bob Balderas

Vice President, Biological Sciences

BD Biosciences

23-15456-00

A

B

CD3 Pacific Blue™

SS

C

CD4 AmCyan

CD

8 A

PC

-Cy7

All cellular events CD3+ gate C

D45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD

45R

A P

E-C

y7

CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5 CD28 PerCP-Cy5.5

CD8+ IFN-γ+ CD8+ IFN-γ+ IL–

2+

CD4+ IFN-γ+ CD4+ IFN-γ+ IL–

2+

SS

C

SS

C

SS

C

SS

C

CD27 APC CD27 APC CD27 APC CD27 APC

IL-2 PE IL-2 PE

IFN

-g F

ITC

IFN

-g F

ITC

CD4– CD8+ gate CD4+ CD8– gate

C

Multicolor Flow

• Scientists choose dyes based on a number

of considerations:

– Configuration of the instrument

– Type of receptors

– Cell type of interest

– Receptor density

– Types of dye-antibody combinations available

– Brightness of the fluorophores

– Spillover value

All Antibodies… All Colors

CD1 x x x x x x x

CD2 x x x x x x x

CD3 x x x x x x x

CD4 x x x x x x x

CD5 x x x x x x x

CD6 x x x x x x x

CD7 x x x x x x x

CD8 x x x x x x x

CDn x x x x x x x

Multicolor Flow Users • The majority of scientists perform 3, 4, 5, and 6-

color applications. They:

– Underutilize the instruments in their labs

– Co-localize too many colors on a single laser

line

– Often do not consider receptor density

• Advanced users

– Need brighter colors across many laser lines

– Incorporate knowledge of receptors and

function

– Are open to new options

Outline • Receptor density assessments

• Reagent and instrument considerations

– BD Horizon™ Brilliant Violet™ fluorochromes

characteristics: • Excitation/emission

• Brightness

• Spillover

• Buffer compatibility

• New BD Horizon™ Brilliant Ultraviolet™

fluorochrome

• An additional method for performing multicolor flow

cytometry

RECEPTOR DENSITY

BD Quantibrite™ Beads to Determine

Antigen Density

# PE

Molecules

Median

Fluorescence

474 913

5,359 10,367

23,843 44,342

62,336 117,857

# PE = 0.53

x MFI

BD Quantibrite Beads

Gate Four

Populations

Determine Relationship of

No. of PE Molecules to MFI

Use MFI of Stained

Sample to Calculate

Ag Density

Antigen Density Protocol

• Stain three different peripheral mononuclear blood cell (PBMC)

donor samples with PE conjugates of each antibody

– CD3, CD4, CD8, CD25, CD27, CD28, CD45RA, CD45RO,

CD122, CD127, CD132, CCR4, CCR7

• Run on BD LSRFortessa™ and BD FACSVerse™ instruments

• Run BD Quantibrite beads to determine the correlation of the

number of PE molecules to median fluorescence intensity (MFI)

values

• Calculate antigen density for each antibody based on the PE

signal in positive cells

Ag Density: CD45RA, CD127, CD132

CD45RO

CD127

CD132

Donor 1 Donor 2 Donor 3

12121 11852 11264 Calculated Ag

Density

288 274 359

1664 1760 2017

PE Fluorescence

T-Cell Antigen Density: BD FACSVerse

BD LSRFortessa

BD FACSVerse

Donor

T-Cell Antigen Density: BD LSRFortessa

BD LSRFortessa

BD FACSVerse

Donor

T-Cell Antigen Density

BD LSRFortessa

BD FACSVerse

Donor

FLUOROPHORE COMPARISON

Fluorophore Comparison Protocol

• Stain three different PBMC donor samples with all

possible conjugates of each antibody

– CD3, CD4, CD8, CD25, CD27, CD28, CD45RA,

CD45RO, CD122, CD127, CD132, CCR4, CCR7

– 16 different fluorophores

– Approximately 400 samples total

• Run on a BD LSRFortessa instrument

• Gate on lymphocytes

• Calculate 95-5 ratio (Cytobank) as a distance metric

between negative and positive cell populations

95-5 Ratio Calculation for Surface Markers

CD4 PE

7.23

95th percentile = 70250

Asinh(70250) = 6.84

5th percentile = -60

Asinh(-60) = -0.39

Asinh(95th percentile) – Asinh(5th percentile)

= 6.84 – (-0.39) = 7.23

The 95-5 ratio is distance between the 95th

and 5th percentiles of data in asinh space.

CD4: 17 Fluorophores Compared

V500

Alexa Fluor® 647

PE

6.71

4.46

3.11

Donor

95-5

ra

tio

Eighteen Fluorophores Compared Across 12

Antigens

Antigen

Donor

Rela

tive B

rig

htn

ess

NEW FLUOROPHORE OPTIONS

Sirigen Polymers Overview

Direct Reporters

Many units = efficient light

harvesting

Properties controlled by size and

composition

>10x brighter than conventional

organic dyes

Replace poor performing reporters

Brightness of QDots and Fluorescent Proteins…with the

well defined and tunable features of dye chemistry

h h h h h

Brilliant Violet Fluorochromes

BV421 BV510 BV605 BV650 BV711 BV786

Brilliant Violet 510 (BV510) is Brighter than

BD Horizon™ V500

BVBV510

Ms CD11 b staining

BV510

V500

Reagent Fluor Stain

Index

Ms

CD11b

BV510 25

V500 10

FITC 25

Hu CD19 BV510 49

V500 17

BV510 is Brighter than Qdot 525

Hu CD4 Biotin + SAV

BV510

Qdot 525

NEW FLUOROCHROME

BRIGHTNESS RANKING

Brilliant Violet Reagents are Bright

BV421 BV711 BV650

BV786 BV605 BV510

PE

Example: CD4 staining, human LWB

PE PE

Brightness Ranking: Stain Index

• Stain index absolute values will depend on:

– Reagent, clone selection

– Instrument configuration (laser wavelength, laser power,

filters)

– Instrument setup

• Ideally, ranking should be established for a specific

instrument

Brightness Ranking

BRIGHT Brilliant Violet™ 421, Brilliant

Violet™ 650, Brilliant Violet™

711, Brilliant Violet™ 786

PE, PE-CF594, PE-Cy™7

APC

MODERATE Brilliant Violet™ 605, BV510

FITC, Alexa Fluor® 488,

PerCP- Cy™5.5

Alexa Fluor® 647, Alexa Fluor®

700

DIM BD Horizon™ V450, V500,

AmCyan

APC-Cy™7/APC-H7

NEW FLUOROCHROME FOR THE

355-NM UV LASER

BD Brilliant UltraViolet™ 395 (BUV395) Spectra

BUV395

BUV711

BUV 395

BUV395

• Excitation maximum of 347 nm, optimum for a 355-nm

ultraviolet laser

• No significant excitation by a 405-nm, 488-nm, 532-nm,

561-nm, or 640-nm laser

• Emission maximum of 395 nm

BUV 395 Brightness

BUFFER COMPATIBILITY

Buffer Compatibility (1)

Brilliant Violet reagents are compatible with:

• EDTA and heparin blood collection tubes

• BD PharmLyse™ and BD FACS™ Lysing Solution

• Paraformaldehyde-based fixatives

• BD Cytofix™ fixation buffer/BD Perm/Wash™ buffer

• BD Pharmingen™ transcription factor buffer set

• BD Phosflow™ perm buffer III

Buffer Compatibility (2): Intracellular Staining

Unstimulated CMV pp65 SEB

CD4

CD8

IL-2 BV421

IFN

-g P

E

IL-2 BV421

IFN

-g P

E

BV 421 Does Not Alter Cell Functionality:

CD107a Staining

Unstimulated CMV pp65 SEB

CD

107a B

V421

IFN-g PE

CD

107a B

V421

IL-2 APC

FIVE FLUOROCHROMES,

MINIMAL COMPENSATION

Five Fluors, Minimal Compensation

• We have now developed five fluorochromes with:

– moderate to high brightness

– minimal or NO spillover between them

• Pre-requisite: 5-laser flow cytometer

No Compensation, Optimal Resolution

Single

stainedl

SI: 129 SI: 201 SI: 72

SI: 84

SI: 127 SI: 200 SI: 65

Mixed

CD3 BV421 CD8 FITC CD4 BUV395

SI: 75

CD45RA APC CD27 PE-CF594

SI: 33

SI: 32

Single

stainedl

Mixed

Example: 5-Color T-Cell Panel

5-Color B-Cell Panel

CD19 BUV395

CD27 BV421

IgD FITC

CD38 PE-CF594

IgM APC

IgM APC

IgD

FIT

C

CD

27 B

V42

1

CD38 PE-CF594

FSC

SS

C

CD19 BUV395

SS

C

IgD

FIT

C

CD27 BV421

Memory

Switch

Naïve

SENTINEL STAINING:

T AND B CELL

Panel Description

I Nat Rev Immunol. 2012 February 17;12(3):191–200.

Sentinel Marker Multicolor Flow

(Normal Cells Follow Rules)

Sentinel Cells T cell B cell NK Monocyte Cell Type 5

Sentinel Marker CD3 CD19 CD56 CD14 Sen Marker 5

color-1 color-6 color-7 color-8 Color-9

color-2 T-cell sub1 B-cell sub1 NK-cell sub1 Mono sub1

color-3 T-cell sub2 B-cell sub2 NK-cell sub2 Mono sub2

color-4 T-cell sub 3 B-cell sub3 NK-cell sub3 Mono sub3

color-5 intracellular 1 intracellular 1 intracellular 1 Intracellular 1

5 color 6 color 7 color 8 color 9 color

5 specificities 10 specificities 15 specificities 20 specificities

25

specificities

This approach could add a tenth color and sixth Sentinel Cell Marker 6, and add

three subsets and one intracellular marker in a sequential manner.

Sentinel Stain Experiment

• Stain three PBMC donor samples with

overlapping panels

• Run on a BD LSRFortessa instrument

• Gate on B or T cells, then proceed with

gating hierarchy

Sentinel Staining Panel

Fluorophore T cells B cells

BV421 CD3

V500 CD45RA

BV605 CD19

FITC CD45RO IgG

PerCP-Cy5.5 CD8 IgM

PE CD25 IgD

PE-Cy7 CD127 CD24

APC CD28 CD38

Alexa Fluor® 700 CD27

APC-H7 CD4 CD20

10 Colors = 16 Antibodies

Sentinel Analysis Strategy: T Cells

Naïve Helper T helper

Central Memory

Effector Memory

Naïve Cytotoxic Central Memory

Effector Memory

Sentinel Analysis Strategy: B Cells

Memory B cells

Sentinel Analysis Workflow

Find major cell subsets based on

sentinel markers

Find subsets within each cell type

Present the population hierarchy in one

figure

Conclusions

• Brilliant Violet dyes offer a wide array of options for

successful multicolor flow cytometry.

• Brightness is a key feature of Brilliant Violet dyes that

enhances resolution.

• Panel design needs to be carefully assessed in order to

mitigate spillover issues and antigen density.

• Brilliant UltraViolet dyes are an exciting and promising

new family of dyes.

Thank You

Cytobank

Nikesh Kotecha

Primity Bio

Peter Krutzik

BD Biosciences

Brent Gaylord

Alan Stall

Maria Jaimes

Cynthia Lane

Trent Colville

Rinku Jana

Jeanne Elia

Larry Duckett

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

APC-Cy7: US Patent 5,714,386

Alexa Fluor is a registered trademark of Life Technologies Corporation.

Cy™ is a trademark of GE Healthcare.

CF™ is a trademark of Biotium, Inc.

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