Providing total solutions to yourimmuno-oncology research workflow
Discover how BD can enable deep and robust data

Table of contents
Blood and tissue collection and dissociation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
BD Vacutainer® Products family. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
BD Horizon™ Dri Tumor and Tissue Dissociation Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Cell staining antibodies, panels, dyes and reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
BD Horizon Brilliant™ Dyes and Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
BD OptiBuild™ Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
BD Horizon™ Dri Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
BD FACSLyric™ Flow Cytometer identifies regulatory T cells with extraordinary sensitivity. . . . . . . . . . . . . . . . . . . . . . . 13
BD Horizon Brilliant™ Dyes and Antibodies enable discovery research for immunotherapy. . . . . . . . . . . . . . . . . . . . . . 14
Flow cytometry, cell sorters and cell analyzers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
BD FACSMelody ™ Cell Sorter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
BD FACSLyric™ Flow Cytometry System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
BD FACSymphony ™ Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Multiomics, scRNAseq, AbSeq, gene panels and multiplexing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Single-cell, end-to-end workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
BD Rhapsody ™ Single-Cell Analysis System.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Data from flow cytometry and multiomic analysis in immuno-oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
BD FACSLyric™ System identification of immune checkpoint receptor expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
BD FACSMelody™ System sorting and assessment of regulatory T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
BD® AbSeq scRNAseq and CAR T cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Multicolor flow cytometry analysis of exhausted T cells.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
BD FACSCelesta™ Flow Cytometer assessment of PD-1 receptor occupancy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
BD Accuri™ Flow Cytometer analysis of immuno-oncology biomarkers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Informatics, flow and RNAseq analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
FlowJo™ and SeqGeq™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Support. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2

| Immuno-oncology

Immuno-oncology |3

Supporting you in designing immuno-oncology assays from discovery researchthrough clinical trials, BD is enabling scientific discoveries across various
immuno-oncology research applications

Instrument platforms



Reagents

At BD, we know that the goal of your research is to transform patient careby developing new cancer therapies. BD will support you in this noble journeyby providing the high-quality tools to generate reliable data every time,
which allows you the ability to advance immuno-oncology research.
Built on a solid foundation of over 40 years of experience in immunology,
we bring unmatched legacy and expertise to help you achieve your goals
and breakthrough new barriers in the field of immuno-oncology.
4| ImmunotherapyImmunology experts

Whether you are studying new checkpoint inhibitors, evaluating multiple
immunotherapy biomarkers, advancing CAR T-cell therapy research,developing new strategies for immune monitoring or discovering new
frontiers in cancer vaccines—we’re here to help simplify the complexity
and guide you on your path from discovery research through clinical trials.

Visit bd.com/Immuno-oncologyIncreased performance for high-quality results: BD aims to provide
a complete solution for your immuno-oncology research workflow

To have meaningful, profound effects
on patient outcomes and quality oflife, you need confidence in the data
you generate. To move from early
research through clinical trials andeventually to the clinic, you needreliable, optimized results with the ability
to transfer high-quality data througheach stage of your research.

Paving a path from discovery to clinical.
BD is dedicated to developing innovative, streamlined
workflow solutions across the spectrum of discovery,translational and clinical research applications.
Bringing quality and reliability tohigh-complexity workflow solutions.
Our instruments, reagents and panels are seamlessly
optimized, producing higher-quality data outputs.

Start with quality specimen collection to achieve better results

The BD Vacutainer® Products family: Cell and biomarker
preservation
Global: Blood cell and biomarker preservation is widely used in clinical and biomarker
research across the world
Flexible: Used with a variety of downstream molecular, multiomic and cellular applications
Reproducible: Helps ensure reproducibility and accuracy of data and workflow
efficiency when measuring biomarkers• Trusted by leading hospitals and research institutions to enhance sample quality,
workflow efficiency and healthcare/laboratory personnel safety• Backed by unrivaled customer support and training

BD Vacutainer® Products

Blood and tissue

Cell staining

Flow cytometry

Multiomics

Informatics

Cat no.

Description

collection anddissociation

antibodies, panels,
dyes and reagents

cell sorters and cell
analyzers

scRNAseq, AbSeq, genepanels and multiplexing

flow and RNAseq
analysis362753

BD Vacutainer CPT™ Mononuclear Cell Preparation Tube—sodium heparin

8.0 mL362760BD Vacutainer CPT™ Mononuclear Cell Preparation Tube—sodium citrate

4.0 mL362761

BD Vacutainer CPT™ Mononuclear Cell Preparation Tube—sodium citrate

8.0 mL

7621165

PAXgene Blood DNA Tube

2.5 mL

762165PAXgene Blood RNA Tube

2.5 mL

Quality matters.
There’s a patient at the end of everything we do.
6| Immuno-oncology

Qty.
®

®

®

®
®

Blood and tissue collection and dissociation |7

BD Horizon™ Dri Tumor and Tissue Dissociation Reagent:
Gentle and effective dissociation with excellent epitope preservation

Excellent performance and preservation: 97% of the 188 cell surface markers were
not compromised by the BD Horizon™ Dri Tumor and Dissociation Reagent
Figure B.

Figure A.
Marker

GentleEffective

Accurate

Maximizes cell yields during
dissociation while minimizing
cell death

Efficiently dissociates a varietyof tumor types to enable
single-cell studies

Maintains the heterogeneityand diversity of your samples

CD1a
CD1b
CD1d
CD2
CD3
CD4
CD4v4
CD5
CD6
CD7
CD8a
CD8b
CD9
CD10
CD11a
CD11b
CD11c
CD13
CD14
CD15
CD15s
CD16
CD18
CD19
CD20
CD21
CD22
CD23
CD24
CD25
CD26
CD27
CD28
CD29

Pop

5
4
4
4
4
4
4
4
4
4
4
1
4
6
6
6
5
5
5
6
5
3
3

6
4
4
4
4
4

SI MFI % pos

Marker

Pop

CD30
CD31
CD32
CD33
CD34
CD35
CD36
CD37
CD38
CD39
CD40
CD43
CD44
CD45
CD45RA
CD45RB
CD45RO
CD46
CD47
CD48
CD49a
CD49b
CD49c
CD49d
CD49e
CD50
CD51/61
CD53
CD54
CD55
CD56
CD57
CD58
CD59

4
5
6
5
6
5
5
5
5
1
4
6
4
3
3
6
1
1
5
2
2
1
4
2
6
5
6
2
6
3
3
4
1

SI MFI % pos

Marker

Pop

CD61
CD62E
CD62L
CD62P
CD63
CD64
CD66 (a-e)
CD66b
CD66f
CD69
CD70
CD71
CD72
CD73
CD74
CD75
CD77
CD79b
CD80
CD81
CD83
CD84
CD85
CD86
CD87
CD88
CD89
CD90
CD91
CDw93
CD94
CD95
CD97
CD98

5

SI MFI % pos

4
2
5
6
6
5
5
1
2
5

4
5
5
5
5
6
6
5
5
6
9
5
5
5

Tumor types evaluated by BD or external investigators include lung, breast, colon,
lymphoma, melanoma/skin, pancreatic, esophageal, kidney, sarcoma and brain.

 Visit bd.com/CDRR or contact your local BD representative to learn how BD Horizon™ Dri Tumor and Tissue Dissociation can support your lab.
8

| Blood and tissue collection and dissociation

Marker

Pop

CD99
CD99R
CD100
CD102
CD103
CD105
CD106
CD107aCD107b
CD108
CD109
CD112
CD114
CD116
CD117
CD118
CD119
CD120aCD121aCD121b
CD122
CD123
CD124
CD126
CD127
CD128b
CD130
CD134
CD135
CD137
CD137L
CD138
CD140aCD140b

5

5
1
2
6
5
4
5
5
2
5
5
2
5
3
2
5
4
5
5

5

2
2

SI MFI % pos

Marker

Pop

CD141
CD142
CD144
CD146
CD147
CD150
CD151
CD152
CD153
CD154
CD158a
CD158b
CD161
CD162
CD163
CD164
CD165
CD166
CD171
CD172b
CD177
CD178
CD180
CD181
CD183
CD184

1
1
2
1
4
1
2
5
3
9
9
5
5
5
5
5
5
5
7
5
6
4
5

CD193
CD195
CD196
CD197
CD200
CD205
CD206
CD209

5

5
5
5

SI MFI % pos

Marker

Pop

CD220
5
CD221
1
CD226
4
CD227
5
CD229
4
CD231
1
CD235a
CD243
CD244
5
CD255
5
CD268
3
CD271
1
CD273
CD274
2
CD275
5
CD278
CD279
CD282
5
CD305
5
CD309
CD314
4
CD321
5
CDw327
3
CDw328
5
CD329
5
CD335
3
CD336
CD337
3
CD338
5
CD340
1
abTCR
4
b2-microglobulin 4
BLTR-16CLIP
3SI MFI % pos

MarkerPop

EGF Receptor
fMLP receptor
gdTCR
HPC
HLA-A,B,C
HLA-A2
HLA-DQ
HLA-DR
HLA-DR, DP, DQ
Invariant NK T
DGD2
MIC A/B
NKB1
SSEA-1
SSEA-4
TRA-1-60
TRA-1-81
Vb 23
Vb 8
CD326
CD49f
CD104
CD120b
CD132
CD201
CD210
CD212
CD267
CD294
CLA
Integrin b7

1
5
4
5
5
5
5
5
5
5
6
2
3

1
1
1
5
5
1
5
5
6
6
10

SI MFI %% pospos

Number of markers affected
% pos

MFI

SI
0

50
No pos expression

100
0–15%

16–30%

150

200

>30%

Figure 1. Preservation of cell-surface epitopes.
(A) Surface marker expression was assessed across the BD catalog
for all 10 distinct cell population. Markers that demonstrated lessthan 20% expression are represented as grey boxes. For eachmarker, a single population (T cells, non-T cells) was chosen for the
analysis. This target population is indicated by a number (1–10)(column heading is “Pop,” see poster abstract for population
assignment numbers). Untreated and treated (exposed to enzyme)
samples were compared using stain index (SI), mean fluorescenceintensity (MFI) of positive signal and the % of the population thatwas clearly positive for a given marker (% pos). Bright blue
indicates a change of less than 15%, light blue indicates a changeof 16%–30% and dark blue indicates a change of 30% or more.
(B) Graphical depiction demonstrating the majority of markers
measured had preserved epitopes measured by MFI and SI,
showing less than 0%–30% change following dissociation.
Middlebrook AJ, Austin C, Santos D, et al. The effects of enzymatic
digestion on epitope detection by flow cytometry [abstract]. In:
Proceedings of the American Association for Cancer Research
Annual Meeting 2018; April, 2018; Chicago, IL. Philadelphia, PA:
AACR; Cancer Res. 2018;78(13 Suppl):Abstract nr 2113.

Ensuring precious samples are preserved right the first time.
Blood and tissue collection and dissociation |9

BD Horizon Brilliant™ Dyes and Antibodies—color your world:
Discover how these innovative dyes can give you confidence in your resultswith improved resolution and flexibility in experimental design

BD OptiBuild™ Reagents provides on-demand access to thousandsof antibody-dye conjugate combinations to enable flexible panel design

Our comprehensive portfolio of over 9,000 immunology andimmuno-oncology related reagents are designed to help
efficiently identify key insights—faster—by enabling
characterization of the cells and biology relevant toyour research

Expand your panel design possibilities
with BD OptiBuild™ On-Demand Reagentsby uncovering more fluorochrome
options on the antibodies you need

Brighter: For rare cells like tumor-infiltrating lymphocytes, or cells that have few
receptors on the surface, bright reagents are essential in distinguishing these dim cells
from others in a sample

Simple and flexible: Whether you want to minimize
compensation or add new markers to complex
experiments, BD OptiBuild™ Reagents provide flexibility toevaluate new colors and simplify your panel design

Pioneering: BD Horizon Brilliant™ Polymer Dyes were developed from advanced Sirigen
dye technology, enabling higher parameter flow cytometry experiments in order tobetter discern populations and garner deeper insights• Access to new antibody-dye combinations made on
demand with rapid turnaround times to help you
meet research deadlines

Reliable: Lot-to-lot consistency and a commitment to high quality ensures better
performance and confidence throughout longitudinal studies• Consistent performance to ensure reliable results

Breaking barriers to discovery starts with the right reagents and panels. BD has an
expansive portfolio of dyes across multiple laser lines with a multitude of antibody
conjugates for each dye—providing you with the choice and flexibility you needto take your research to the next level.

• Come in convenient 50-μg vials

Fast and convenient: Unlike traditional large-scale,expensive custom conjugates, these reagents:• Can be ordered as a catalog reagent for optimal
panel design• Are made on demand, and usually ship in lessthan 72 hours** Within U.S., Canada and Europe

Visit bd.com/Optibuild
10 | Cell staining antibodies, panels, dyes and reagentsCell staining antibodies, panels, dyes and reagents | 11

Regulatory T-cell panel: Identification of Treg subsets and understanding Treg
heterogeneity on the BD FACSLyric™ Flow Cytometer

Predesigned BD Horizon™ Dri Panels: Dried reagent cocktails for improved
workflow efficiency and consistency
Minimize time spent on cocktailing reagents and reduce day-to-day variability

BD Horizon™ Dri Memory T-Cell Panel

Th17/Th22-like

CD183 BV711

CD196 APC-R700

CD4+ Tconv cells

CD45RA- Tconv cells

CD183- Tconv cells

RTE

CD31 BV605

CD45RA PE-Cy7

SSC

Th2-like

CD45RA PE-Cy7

Lyse, wash and analyze by
flow cytometry

BD Horizon™ Dri TBNK+CD20 Reagent Panel

Fluorochrome

Marker

Clone

Fluorochrome

Marker

Clone

Fluorochrome

Marker

Clone

FITC

CD16

3G8

FITC

CD197

150503

BD Horizon Brilliant™ Violet 450

CD20

L27

PE

HLA-DR

L243

PE-Cy™7

CD95

DX2

FITC

CD3

SK7

PerCP

CD14

MφP9

BD Horizon™ APC-R700

CD27

M-T271

PE

CD16

B73.1

APC

CD192 (CCR2)

LS132.1D9

APC-H7

CD3

SK7

PE

CD56

NCAM16.2

BD Horizon™ V450

CD4

SK3

PerCP-Cy™5.5

CD45

2D1 (HLe-1)

BD Horizon™ V500-C

CD8

SK1

APC

CD19

SJ25C1

BD Horizon Brilliant™ Violet 605

CD45RA

HI100

PE-Cy™7

CD4

SK3

APC-Cy™7

CD8

SK1

In addition to predesigned panels, BD Custom Technology Team (CTT) offers contract manufacturing of multicolor panels in lyophilized, liquid and/or dried formats to minimize
the error(s) and time associated with manual cocktailing of reagents, increase the reagent stability, and significantly enhance performance consistency.

CD45RA-

CD45RA PE-Cy7

Th1

CD183 BV711

CD194 BV786

Add sample, vortex
and incubate

CD45RA PE-Cy7

BD Horizon™
Dri Panel Reagent

CD45RA- CD45RA+

Th1-like

CD45RA+ Tregs

CD183- Tregs

CD45RA- Tregs

CD45RA PE-Cy7



SSC



*Versus liquid reagents

Stability*: Improved performance and shelf life overliquid cocktails with the advantage of room-temperaturestorage. Offers a performance guarantee for one year
at room temperature.

BD Horizon™ Dri Monoset Panel

Improve ease-of-use and reduce time spent
preparing reagent cocktails

CD4+ Tregs

Lot-to-lot consistency*: High-quality reagents and BD
technology built into a reagent panel. Long-term
reagent stability further enhances assay reproducibility.

Convenience*: Ready-to-use, dried-down, single-use
tubes offering workflow efficiency while providing
greater accessibility and convenience. Simply resuspend
the dried reagents and add your sample, with no needfor cocktailing.

Conventional CD4+ T cells

Easy-to-use*: All-in-one, multicolor panels optimized forcell characterization can be used across labs and sites toenable reliable results.

FoxP3+ Tregs

CD194 BV786

BD offers predesigned (consensus), performance-optimized panels in dried down, ready-to-use multicolor panels optimized and tested for memory T-cell, monocyte subset
and TBNK cell characterization.

Th2

Th17/Th22

Regulatory T-cell panel
7-color Treg panel

Additional Treg homing drop-ins

CD127

FoxP3

CD15s

CD31

CD161

CD183

CD4

CD194

CD45RA

CD196

CD25
CD3

CD196 APC-R700

Figure 2. Demonstration of modular flow cytometry approach (7 + 5 colors) to characterize Tregs.
The 7-color backbone panel was used to identify Tregs and was supplemented with a homing panel whose
expression correlates with different aspects of Treg biology, such as homing, function, maturation,
proliferation, trafficking and stability. The homing panel successfully identified 3 subpopulations of Th-likeTregs and recent thymic emigrants within the Treg population (upper panel). Conventional T-helper cell subsets
Th1, Th2 and Th17/Th22 were also identified within the conventional CD4+ T cells (Tconv) (lower panel).
Corselli M, et al. A modular, multicolor approach to regulatory T-cell characterization. White paper.
BD Biosciences, San Diego, CA. 2018.

High-resolution identification of cell populations makes it an ideal solution
for expanded immunophenotyping of immuno-oncology-relevant cell populations.

 Visit bd.com/CDRR and bd.com/CustomPanels or contact your local BD representative to learn how BD Horizon™ Dri Panels and custom panels can support yourlab.

12 | Cell staining antibodies, panels, dyes and reagents

Cell staining antibodies, panels, dyes and reagents | 13

BD Horizon Brilliant™ Dyes and Antibodies are enabling
discovery research for immunotherapy
Minimize your time spent troubleshooting and maximize your impact

Cat no.

Markers

Fluorochrome/Dye

Cat no.

Markers

Fluorochrome/Dye

See mouse dump
table below

Mouse dump

FITC

See mouse dump
table below

Mouse dump

FITC

See mouse dump
table below

Mouse dump

FITC

560662CD8PerCP-Cy™5.5

560635

CD195

PerCP-Cy™5.5

560835CD3PerCP-Cy™5.5

PE560933CD163

PE555413

CD19

PE

PE-CF594

564063

CD206

PE-CF594

562393

CD11c

PE-CF594

560684

CD28

PE Cy™7

557743

CD11b

PE Cy™7562101

NKp46

PE Cy™7555342

CD3

APC561306

CD16

APC561304

CD16

APC

CD45

APC-H7560180CD14

APC-H7560180CD14

APC-H7CD4

AF700560566

CD45

AF700

560566

CD45

AF700

562885

CD45RA

BV421

564067

CD192

BV421

562518IgD

BV421

Live/Dead cells

FVS 510564406

Live/Dead cells

FVS 510564406

Live/Dead cells

562845

HLA-DR

BV605

562845

HLA-DR

BV605

563159

CD25

BV711

563171

CD33

BV711

563161

CD123

BV711

563324

CD127

BV786

563383

CD15

BV786

564058

CD56

BV786

561908DNA

Hoechst 33342

561908DNA

Hoechst 33342

561908DNA

Hoechst 33342

14 | Cell staining antibodies, panels, dyes and reagents

553079mCD45553592mH2Kd561032

mTer119558738mCD31561849mCD41553266

mCD71

10

5
0
30
20
10
0

0
40

20

BV605

Antibody

20

30

HLA-DRCat no.

30

FVS 510562846

Mouse dump

0

10

10
0
NSG

SGM3

NSG

Mouse strain

SGM3

10.0
7.5
5.0
2.5
0
40
30
20
10
0
30
20
10
0
NSG

20
CD8+ naive

56440620

CD4+ central memory557922

40

15

CD8+

56027420

31

CD4+ naive

CD197

80

CD4+ naive

562381

0

Tregs

CD38

10

0

60

10

20

25

CD4+

55548050

Percent of CD45 positive

Fluorochrome/Dye30

31

CD8+ effector memory

Markers

40

75
T cellCat no.

100

Percent of CD45 positive

NK/DC/B-cell panel

1031CD8+ central memory

Myeloid panel10

Age of mice in weeks

CD4+ effector memory

T-cell panel

Age of mice in weeks

Age of mice in weeks

Percent of CD45 positive

Perform a comprehensive and detailed analysis of the immune system with optimized flow cytometry panels todetermine which strains are a suitable host for patient-derived xenografts (PDX).

15
10
5
0
NSG

SGM3

NSG

SGM3

SGM3

NSG

SGM3

Mouse strain

Tissue:Bone marrow

PBMC

SpleenPBMC (human reference)

Mouse strain

Figure 3. Sample data from T-cell panel analysis. 14-color flow cytometry panels were used on immunodeficient mice NOD scid gamma (NSG™) and triple transgenic NSG™ mice expressing human cytokines KITLG,CSF2 and IL-3 (NSG™-SGM3) and humanized by transplantation of human cord-blood-derived CD34+ hematopoetic stem cells. Sample tissues (bone marrow, spleen and peripheral blood) were stained with a 14-color
antibody panel designed to enumerate T-cell subsets. The box plots show the reconstitution of various T-cell subsets in 2 mouse models at 10 and 31 weeks of age. The green bands across each plot represent the rangeof each subpopulation in peripheral blood as measured in normal healthy adult donors (n = 6) using the same panel. Mouse model and data generated in conjunction with The Jackson Laboratory.
Middlebrook AJ, et al. Comprehensive evaluation of human immune system reconstitution in NSG™ and NSG™ -SGM3 mouse models toward the development of a novel ONCO-HU™ xenograft model. BD Biosciences and
The Jackson Laboratory white paper. BD Biosciences, San Jose, CA; BD Technologies, Raleigh-Durham, NC; and The Jackson Laboratory, Bar Harbor, ME. 2017.

Cell staining antibodies, panels, dyes and reagents | 15

BD FACSMelody™ Cell Sorter provides
simple, fast and quality
cell sorting ideal for enrichmentof rare populations

BD FACSLyric™ Flow Cytometry Systemis helping drive reproducibility
and standardization

Making the complex world of flow
cytometry and sorting more accessible,
enabling deep scientific insights withexcellent results
Fast and efficient: Truly simple, easy to learn and maintain cell sorting. Automation
enables the system to be ready in less than 17 minutes, maximizing uptime and ability
to run samples sooner
Versatile: Perfect for sorting populations of cells into tubes or plates before flow
cytometry analysis and scRNAseq
High performance: Excellent sensitivity for accurate results for low-density cellmarkers and high throughput for rare cell collection
Proven technology: Core technology derived from the highly cited BD FACSAria™

Trusted partner: BD, a leader in flow cytometry,
continues to improve instrumentation, software andreagent solutions to help you achieve your research goals
Standardized: Easy to transfer between CROs, othermanufacturing sites and collaborators with reproducible
and accurate performance
Portability: Strengthen partnerships and expand global
collaborations through assay portability and sharing

Attain deeper insights faster with the customizable, high parameter
BD FACSymphony™ Cell Analyzer

Deep insight: Simultaneous measurement of up to
50 different characteristics of a single cell
Sensitivity: Improved detection systems enable you
to identify and analyze rare cell types and events
Expandable: Configured to meet the requirementsof your specific research application needs today withroom for growth tomorrow

User-defined assayCreateuser-developed assayExportuser-developed assay



Send

to collaborators

Importuser-developed assay

Cell Sorter

Better performance for better results. We’ve done the hard work for you.
 Visit bd.com/FACSMelodySorter and bd.com/FACSLyric
16 | Flow cytometry, cell sorters and cell analyzers

Customization. High-parameter flow cytometry to fit your needs.
Visit bd.com/FACSymphony

Flow cytometry, cell sorters and cell analyzers | 17

Single-cell, end-to-end workflow with informatics
increases the experimental power for your research

BD® AbSeq Antibody-Oligo
Conjugates
Choose from a menu
of high-quality
BD antibody-oligos
to detect cell surface
proteins simultaneouslywith mRNA-sequencing
for multiomic analysis

BD® Single-Cell
Multiplexing Kit
Pool up to 12 samples
to minimize batch
effects and reduce time
to discovery andexperimental costs

BD Rhapsody™
Express System
Single-cell capture,bead-based mRNA
isolation andbead retrieval

BD Rhapsody™ Targeted
Single-Cell RNA-Seq Panels
Single-cell capture,bead-based mRNA
isolation andbead retrieval

The BD Rhapsody™ Single-Cell Analysis System with a complete setof multiomic tools, including reagents and analysis software,
helps empower and streamline your research with a targeted approach
Next-generation
sequencing• Immune response
panel (human)• Immune response
panel (mouse)•O nco-BC panel(breast cancer)• T-cell panel• Custom panels

The BD Rhapsody™ Whole
Transcriptome Analysis(WTA) Amplification Kit
Identify genes of interest
that can be used for building
your targeted panels which
can be used in conjunction
with BD ® AbSeq• Supplementary panels

BD Rhapsody™
Single-Cell Analysis System
Single-cell capture,bead-based mRNA
isolation and beadretrieval with imaging
and cell workflow QC

For over 40 years, BD has been a trusted partner in single-cell biology.
You can rely on us to help open new frontiers in single-cell analysis.
18 | Multiomics, scRNAseq, AbSeq, gene panels and multiplexing

BD Bioinformatics• Alignment and mapping
primary analysis software• Easy-to-use SeqGeq™
v1.6 Secondary Analysis
Software for clusteringvisualization andelimination of multiple
data for clean differential
gene expression

Achieve results while saving time and money with assaysthat dramatically reduce experimental cost and complexity,
improving data and increasing efficiency of sequencing
Comprehensive: A broad multiomic solution for single-cell analysis
Synergistic: BD® AbSeq enables synergies with high-parameter flow cytometry,allowing discoveries with BD® AbSeq to transfer to panel design and WTA discoveriestransfer to targeted panels with BD® AbSeq
Resolution: BD® AbSeq, when paired with WTA and mRNA targeted panels, allows
for enhanced cell clustering and resolves differences between RNA and protein
expression levels“BD provides a complete range of key
technologies and deep expertise to facilitate
discoveries in immuno-oncology, withexpanded focus on solid tumors. These
include efficient single-cell dissociationfrom tumor tissues, the ability to generatelots of usable data from low cell numbers
via high-dimensional flow cytometry or
single-cell RNA sequencing, and new
exciting technologies such as AbSeq.”
Peter P. Lee, MD, City of Hope Comprehensive Cancer Center, Duarte, CA

Visit bd.com/Multiomics

Multiomics, scRNAseq, AbSeq, gene panels and multiplexing | 19

BD FACSMelody™ Cell Sorting System provided sorting and cell isolation
to enable downstream functional assessment of regulatory T cells

CD274 FITC-A

HLA-DR BV510-A

CD3 PerCP-Cy5.5-A

HLA-DR BV510-A

HLA-DR

HLA-DR BV510-A

CD3 PerCP-Cy5.5-A

CD3 PerCP-Cy5.5-A
CD152 BV421-A

CD152 BV421-A

CD152 (CTLA-4)

CD279 BV605-A

CD279 BV605-A

CD279 BV605-A

Expression of immune checkpoint receptors on
peripheral blood immune cells using a 10-color assay on
the BD FACSLyric™ Flow Cytometer. Application note.
BD Biosciences, San Jose, CA. 2017.
Targetantigen

Alternate
name

Clone

Fluorophore

Cat. no.

CD45

PTPRC

2D1

APC-H7560178

CD3

n/a

SK7

PerCP-Cy™5.5

HLA-DR

n/a

G46-6

CD28

n/a

CD134

OX40

CD137

B

CD25 APC

CD25 APCC

CD25 APC

Figure 5. Sorting Treg and Tresp populations. CD4+ T cells wereenriched using the BD IMag™ Human CD4 T Lymphocyte Enrichment
Set-DM and stained using the Treg panel. Dead (7-AAD+) and otherlineage-positive cells (i.e., those expressing CD8, CD14 or CD19) were
excluded, and CD4+ T cells were identified as CD3+ CD4+ (not shown).
(A) Representative final sorting gates of Tregs identified as CD25
high/+ CD127 low/− and responder T cells (Tresps) as CD25 low/−CD127
high/+ are shown within CD4+ T cells. Sorted Tregs (B) and Tresps(C) had greater than 98% purity within the CD4+ T cell gate.
Sorting and downstream functional assessmentof regulatory T cells isolating live cells with the
BD FACSMelody™ Cell Sorter. BD Biosciences
datasheet. BD Biosciences, San Jose, CA. 2018.

BD FACSMelody ™ Cell Sorting System
Cat no.

Markers

Fluorochrome

340949641397

CD3

APC-H7

BD Horizon™ BV510

563083563877

CD4

BV786

CD28.2PE-Cy™7

560684340939

CD25

APC

ACT35

PE

555838557938

CD127

PE

4-1BB

4B4-1

APC

550890565310CD8PerCP-Cy ™5

CD274

PD-L1

M1H1

FITC

558065562692CD14PerCP-Cy ™5

CD86

B7-2

2331

Alexa Fluor® 700

561124561295

CD19

PerCP-Cy ™5

CD152

CTLA-4

BNI3

BD Horizon™ BV421

562743

CD279

PD-1

EH12.1

BD Horizon™ BV605

563245559925

Dead cells

7-AAD

Helping researchers characterize and quantify immune checkpoint
receptors based on decades of deep immunology expertise.
20 | Data from flow cytometry and multiomic analysis in immuno-oncology

A

CD127 PE

Figure 4. Demonstration of immune checkpoint
receptor expression on activated T cells that are
regulated in part by time-in-culture or by stimulatory
conditions using BD FACSLyric™ Analyzer.
Intermediate concentrations (50 ng/mL) of PMA plusionomycin appeared to induce robust upregulation ofCD134, CD137, PD-L1/CD274, HLA-DR, CD86, CD152
and PD-1/CD279 in CD3+ T cells.

CD127 PE

CD3 PerCP-Cy5.5-A
CD86 Alexa 700-A

CD86 (B7-2)

CD279 (PD-1)
CD3 PerCP-Cy5.5-A

CD3 PerCP-Cy5.5-A

SSC-A

CD3 PerCP-Cy5.5-A

CD274 (PD-L1)

CD86 Alexa 700-A

CD152 BV421-A

CD3 PerCP-Cy5.5-A
CD274 FITC-A

12-hour
PMA (50 ng/mL)
Ionomycin (500 ng/mL)

CD3 PerCP-Cy5.5-A

CD137 APC-A

CD3 PerCP-Cy5.5-A

CD3 PerCP-Cy5.5-A

CD3 PerCP-Cy5.5-A
CD274 FITC-A

CD86 Alexa 700-A

CD3 PerCP-Cy5.5-A

CD137 APC-A

CD137 (4-1BB)

12-hour
PMA (5 ng/mL)
Ionomycin (500 ng/mL)

CD3 PerCP-Cy5.5-A

CD3 PerCP-Cy5.5-A
CD134 PE-A

CD3 PerCP-Cy5.5-A

CD3 PerCP-Cy5.5-A

CD3 PerCP-Cy5.5-A
CD137 APC-A

CD3

SSC-A
CD45 APC-H7-A

CD3 PerCP-Cy5.5-A
CD134 PE-A

CD134 (OX40)

CD134 PE-A

12-hour
Unstimulated

CD3

CD134 PE-A

12-hour
PMA (50 ng/mL)
Ionomycin (500 ng/mL)

CD3 PerCP-Cy5.5-A

CD3 PerCP-Cy5.5-A

SSC-A

12-hour
PMA (5 ng/mL)
Ionomycin (500 ng/mL)

CD3 PerCP-Cy5.5-A

12-hour
Unstimulated

CD127 PE

The BD FACSLyric™ Flow Cytometry System identified
immune checkpoint receptor expression on activated T cells

Whether you choose one rare cell or a whole population, the BD FACSMelody™ Cell Sorter
makes it easy to sort as part of your immuno-oncology workflow.
Data from flow cytometry and multiomic analysis in immuno-oncology | 21

3047 cells (100.0%)603514 mols (5.8%)

30

2

0

1.5

-10

1

-20

0.5

-30
-40
-30

-20

-10

0
Coord 1

10

20

30

0

Coord 1

10

20

30

-40
-30

2987 cells (98.0%)
23636 mols (0.2%)

2

10

1.5

0
1

-10
-20

0.5

-30
-40
-30

-20

-10

0
Coord 1

10

20

30

40

2.5

0

-20

-10

0

Coord 1

10

20

30

MK167 (AbSeq + mRNA)
493 cells (16.2%)
692 mols (0.0%)

30

-40
-30

0.9

40

0.8

30

0.7

20

0.5

0

0.4

-20

0.2

-20

-30

0.1

-30

-40
-30

0

-40
-30

10

20

30

20

30

747 cells (24.5%)
1597 mols (0.0%)

1.2

1

0.8

0
-10

0
Coord 1

10

10

0.3

-10

0

Coord 1

TOPA (AbSeq + mRNA)

-10

-20

-10

20

0.6

10

-20

0.6

0.4

0.2

-20

-10

0
Coord 1

10

20

30

TSCMs are identified as a minor subset within the CAR 19 T cell productand these are found expanded in the blood of patients up to 100 days
post CAR 19 T cell post-infusion (data not shown, see poster for details).
Cai CH, McGuire H, Clancy L, et al. Characterising the functionaland genomic profiles of CAR CD19 T cells using single-cell analyses.
Poster presented at ECI 2018; September 2-5, 2018; Amsterdam,
The Netherlands.

0

BD AbSeq resolved with higher accuracy the transcriptional and surface
phenotype of CAR T cells in the infusion product than scRNAseq alone.
®

22 | Data from flow cytometry and multiomic analysis in immuno-oncology

35%

3%

0%

17.1%

CD279 (PD-1) PE-Cy7

1.5%

6.7%

21.1%

TIGIT BV421
99.8%

CD366 (TIM-3) BV786

0.1%

3.4%

18.8%

CD152 (CTLA-4) PE

53.4%

0.5%

CD279 (PD-1) PE-Cy7

C

81.4%

59%

37.5%

CD223 (LAG-3) AF647

0.1%

CD272 (BTLA) BV605

2%
CD223 (LAG-3) AF647

A

CD272 (BTLA) BV605

Coord 2-30

0log 10 (Number of molecules per cell)-30

0CD45RA (mRNA + AbSeq)

20

Coord 2

10

0

30

2.5

20

Coord 240
3

log 10 (Number of molecules per cell)

40

-10

1.5

79.4%

14.7%

2.6%

CD366 (TIM-3) BV786

0.9%
CD152 (CTLA-4) PE

D

17.1%

12.5%

81.3%

0.6%

62%

8.4%

18%

0.1%

0.1%

0%

CD279 (PD-1) PE-Cy7

99.7%

CD366 (TIM-3) BV786

0.2%
CD152 (CTLA-4) PE

Multicolor flow cytometry
Cat. no.

1.3%

33.3%

5.4%

83.1%

5.1%

60.3%

48.7%

31.4%

CD223 (LAG-3) AF647

CD8 (mRNA + AbSeq)

-20

0.1

16.5%

CD272 (BTLA) BV605

-30
-30

30

2

-20

B

45.5%

TIGIT BV421

20

-10

0.2

-20

A

CD223 (LAG-3) AF647

Coord 1

10

-10

2.5

0

Figure 6. BD® AbSeq characterizing CAR CD19 T cells. Participantsfrom a trial at Westmead Hospital (Sydney, Australia) using CAR T cells
based on 4-1BB coreceptor (instead of CD28) and PiggyBac™ System
for gene modification. Development of a single-cell multiomics
approach was used to study the molecular, functional and transcriptomic
profile of CAR 19 T cells in the preinfusion product and the blood ofpatients following adoptive transfer. This was to test the hypothesis thatsurvival of CAR 19 T cells postinfusion is driven by long-term stem
memory T cells (TSCMs).

CD272 (BTLA) BV605

0

0.3

3

10

log 10 (Number of molecules per cell)-1003048 cells (100.0%)1430977 mols (13.7%)

20

0.4

10

0.5

-20

0.5

0.5

20

CD38 (AbSeq + mRNA)

30

Coord 2

-20

1

-10

40

0.6

log 10 (Number of molecules per cell)1

136 cells (4.5%)
143 mols (0.0%)

log 10 (Number of molecules per cell)

-10

1.5

0

FAS (AbSeq + mRNA)

30

Coord 21.5

2

log 10 (Number of molecules per cell)

0

40

2.5

10
Coord 22

-202987 cells (98.0%)
23636 mols (0.2%)

20

2.5

10
Coord 2

log 10 (Number of molecules per cell)

20

-30
-30

CD45RA (mRNA)

3

Coord 2303047 cells (100.0%)603514 mols (5.8%)

log 10 (Number of molecules per cell)CD8 (mRNA)

30

Multicolor flow cytometry with the BD FACSCelesta™ Flow Cytometer
demonstrated comprehensive immunophenotypic analysis of exhausted T cells

TIGIT BV421®

TIGIT BV421

BD AbSeq scRNAseq and protein expression levels identified T-cell subsets
and rare populations of memory T cells in CAR-T infusion products

0.4%

CD279 (PD-1) PE-Cy7

11.1%

15%

CD366 (TIM-3) BV786

4.9%
CD152 (CTLA-4) PE

Multicolor flow cytometry

Markers

Fluorochrome

Cat. no.

Markers

Fluorochrome

560835CD3 or live/dead cells

PerCP-Cy™5.5

747844

TIGIT

BV421

559925CD3 or live/dead cells

7-AAD

555853

CD152 (CTLA-4)

PE564975

CD4APC-R700

565716

CD223 (LAG-3)

Alexa Fluor® 647

560179CD8APC-H7743986

CD272 (BTLA)

BV605

563963

CD45RA

BV650

561272

CD279 (PD-1)

PE-Cy™7561271

CD197 (CCR7)

FITC742857

CD366 (TIM-3)

BV786

746675

CD95

BV480

Figure 7. Coexpression patterns of inhibitory receptors inunstimulated and in vitro stimulated CD8+ and CD4+ T cells.
The use of bivariate plots enabled the identification of complex
coexpression patterns of inhibitory receptors and highlights theheterogeneous phenotype of in vitro persistently stimulated T cells
and immunophenotypic analysis of exhausted T cells. Plot analysis
was performed to identify subsets of total CD8+ T and CD4+ cells
coexpressing inhibitory receptors within fresh, unstimulated PBMCs
and T cells persistently stimulated in vitro with Dynabeads® Human
Activator CD3/CD28 and human recombinant IL-2 for 9 days.
(A) Bivariate plot analysis provided information on the heterogeneityof CD8+ T cells coexpressing inhibitory receptors. For example, distinct
subsets of cells expressing only TIGIT, only PD-1 or coexpressing both
inhibitory receptors were detected. (B) More complex patterns ofexpression were observed upon persistent stimulation in vitro that
resulted in differential regulation of the inhibitory receptors tested.
For example, while the overall percentage of CD8+TIGIT+ cells
decreased, an increase in cells coexpressing PD-1 and TIGIT was
observed. Interestingly, only a small, discrete subset of CD8+ cells
upregulated CTLA-4 expression, thus confirming the heterogeneityof cells persistently stimulated. (C–D) Similar observations were madefor CD4+ T-cell subsets.
Evaluating the expression patterns of multiple inhibitory receptors
associated with T-cell exhaustion using multicolor flow cytometry.
BD Biosciences white paper. BD Biosciences, San Jose, CA. 2018.

Flow cytometry analysis of multiple T-cell inhibitory receptors can provide deep insight
into T-cell exhaustion.
Data from flow cytometry and multiomic analysis in immuno-oncology | 23

The BD Accuri™ C6 Plus Personal Flow Cytometer demonstrated stimulationof immature dendritic cells, which resulted in both cell maturation andinduction of PD-L1

A high-throughput flow cytometry assay to assess PD-1 receptor occupancy. BD Biosciences datasheet.
BD Biosciences, San Jose, CA. 2018.

10 5

10 6

10 7.210 2.5 10 3

10 4

10 5

10 6

10 7.2

10 7.210 5

10 6+ 24h PBMC supernatantanti-IFN-γ and anti-TNF
Q1-UL
Q1-UR
3.1%
5.8%

10 3

10 4

10 5
10 3

Q1-LR
46.3%

Q1-LL
0.1%

D

Q1-LR
9.9%

Q1-LL
0.5%

10 2.5 10 3

10 4

10 5

10 6

10 7.2

10 1.510 4+ 24h PBMC supernatant
Q1-UL
Q1-UR
0.4%
89.2%

10 4

10 5
10 3

10 4

10 5

CD83 APC

10 4
10 2.5 10 3

10 7.2

Q1-LR
4.1%

C

10 6

Q1-LL
93.2%

10 7.2

Q1-UR
1.4%+ rhIFN-γ and rhTNF
Q1-UL
Q1-UR
0.1%
53.4%

10 6

Q1-UL
1.3%

10 6

10 7.2B

Immature DC

10 1.5Figure 8. Assessment of PD-1 receptor occupancy in vitro after pembrolizumab or nivolumab treatment.
PBMCs from one donor were stimulated overnight with 10 μg/mL of immobilized BD Pharmingen™ NA/LE
Anti-Human CD3. PD-1 expression was assessed on the BD FACSCelesta™ Flow Cytometer and thennivolumab, pembrolizumab or human IgG4 isotype control was added to the cultures. The cells were cultured
with the blocking anti-PD-1 antibodies as well as immobilized anti-CD3 and 10 ng/mL of BD Pharmingen™
Recombinant Human IL-2 for 3 days. The cells were washed and stained with BD Horizon™ BV510 Mouse
Anti-Human CD3 and PE-EH12.1.

(A) Immature DCs obtained on day 6 of culture did not express CD83
(BD Pharmingen™ APC Mouse Anti-Human CD83) or PD-L1 surface
markers. (B–C) Upregulation of these markers was observed uponmaturation induced by 3-day stimulation with rhIFN-γ and rhTNF or
conditioned medium (24h supernatant) from activated PBMC
cultures. (D) Cells were also stimulated with the same conditioned
medium in the presence of 10 μg/mL BD Pharmingen™ Purified NA/LE
Mouse Anti-Human IFN-γ (cat. no. 554698) and 10 μg/mL of TNF(cat. no. 554508) blocking antibodies, which resulted in a significant
inhibition of maturation, as indicated by the lack of CD38 andPD-L1 expression.

A

10 1.5Figure 9. PD-L1 and CD83 expression following induction andblockage of DC maturation. Human monocyte-derived DCs weredifferentiated using 50 ng/mL of BD Pharmingen™ rhGM-CSF(cat. no. 550068) and 50 ng/mL BD Pharmingen™ rhIL-4 (cat. no. 554605).

10 3

®

10 1.5

BD FACSCelesta™ Flow Cytometer with a BD High Throughput Sampler (HTS)
option helped assess PD-1 receptor occupancy

Q1-LR
8.2%

Q1-LL
82.9%

10 2.5 10 3

10 4

10 5

10 6

10 7.2

CD274 (PD-L1) BB515

Analysis of immuno-oncology biomarkers using personal flow
cytometry. BD Biosciences datasheet. BD Biosciences, San
Jose, CA. 2018.

This demonstrated that stimulation of immature dendritic
cells (involved in tumor antigen presentation) with either
conditioned medium (24h supernatant, plot C) or
recombinant cytokines (hIFN-γ and rhTNF, plot B)
resulted in both cell maturation (demonstrated byupregulation of CD83) and induction of PD-L1. However,adding anti-IFN-γ and anti-TNF blocking antibodies to theconditioned medium prevented upregulation of CD83
and PD-L1, and ultimately maturation of the DCs (plot D).

Our flow cytometers can provide robust analysis of PD-1 and other checkpoint
receptor expression on T-cell subsets and help with several high throughput
drug-discovery applications.
24 | Data from flow cytometry and multiomic analysis in immuno-oncology

Several immuno-oncology biomarkers and applications can be investigated with theeasy to use, simple to maintain and affordable BD Accuri™ C6 Flow Cytometer.
Data from flow cytometry and multiomic analysis in immuno-oncology | 25

BD offers a variety of powerful software applications to help you quickly andeasily analyze your data
Flow cytometry analysis: FlowJo™ is the leading
platform for single-cell flow cytometry analysis thathelps your research stand out. With the release of
FlowJo™ v10.6, you can take your analysis to thenext level with new features including spectral
compensation, kinetic overlays, improved
BD FACSDiva™ support and more.

FlowJo™

SeqGeq™

RNAseq analysis: SeqGeq™ v1.6 is a desktop
bioinformatics platform that makes complex scRNA
seq analysis accessible with an intuitive interface.
SeqGeq™ lets you control your analysis—no morewriting R scripts to visualize your data—and easily
share your results for publication and collaboration.
Explore, visualize and shape your next-gen sequencing
data with interactive graphs that are designed to help
you focus on the insights that matter most.

Contact your BD representative today to discuss how these innovative
reagents, instruments and software solutions can provide increased
reliability and quality in your workflow and enable breakthroughscientific insights.
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26 | Informatics, flow and RNAseq analysis

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