BD Flow Cytometry Reagents
Multicolor Panels Designed for Optimal Resolution
with the BD LSRFortessa™ X-20 Cell Analyzer

Proper multicolor panel design takes into account fluorochrome brightness, antigen density,
co-expression, and spillover in the context of a given instrument configuration. The following
experiments were designed to maximize resolution of a variety of cell types on a BD LSRFortessa™
X-20 cell analyzer.
BD LSRFortessa™ X-20
Configuration

Laser

Ultraviolet(355 nm)Violet(405 nm)

FluorochromeFilterBD Horizon™ BUV395379/28

BD Horizon™ BUV496515/30

BD Horizon™ BUV661

670/25

BD Horizon™ BUV737

740/35

BD Horizon™ BV421

450/50

BD Horizon™ BV510

525/50

BD Horizon™ BV605

610/20

BD Horizon™ BV650

670/30

BD Horizon™ BV711

710/50

BD Horizon™ BV786

780/60

BD Horizon™ BB515
FITC
Blue
(488 nm)Yellow-Green(561 nm)

Alexa Fluor® 488

PerCP-Cy™5.5

670/30PE586/15

BD Horizon™ PE-CF594

610/20PE-Cy™7

780/60APC
Alexa Fluor® 647
Red(640 nm)

530/30

BD Horizon™ Fixable
Viability Stain 520

BD Horizon™ APC-R700
Alexa Fluor® 700
APC-H7
APC-Cy7670/30
730/45780/60Similar configurations exist on the following instruments: BD LSRFortessa™
cell analyzer, BD FACSAria™ Fusion cell sorter, and BD FACSAria™ III cellsorter.

Page 2
BD Flow Cytometry Reagents
Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer

B-Cell Panel

Specificity

Clone

Fluorochrome

PurposeCat. No.

CD19

HIB19

BD Horizon™ BUV496

B cells

561295IgM

G20-127PE

B-cell subset

555783IgD

IA6-2

BD Horizon™ BUV395

B-cell subset

563813

CD24ML5

BD Horizon™ BV510

Memory/naïve subset

563035

CD27

M-T271

BD Horizon™ BV421

Memory subset

562513CD20

2H7

APC-H7

Plasmablast subset

560734CD38

HIT2PerCP-Cy™5.5

Plasmablast subset

551400Viabilityn/a

BD Horizon™ Fixable
Viability Stain 520

Cellular viability

564407

ProtocolPBMCs were prepared by using Ficoll-Paque™ Plus according to themanufacturer’s directions and incubated with antibodies in the presence of BD
Horizon™ Brilliant Stain Buffer at room temperature protected from light for 20
minutes and washed prior to acquisition.

DataAnalysis of Human B-Cell Subsets

105Switched Memory-180

CD380102

103

IgD

104

105

105

MemoryTransitional

101102103

CD24104
103

B Cells-430-102 0 102

103

CD38104

105

104

105

IgD

104

105

B Cells

103

103

102

CD190102-1800-0102

FVS520104

105

B Cells-580102

103

IgM

-430

-98

0Naive-102 0102

103
102

CD27

Plasmablasts

104

UnswitchedMemory103

Switched Memory

104

105

B Cells-472-102 0 102

103

CD20104

105

Page 3
BD Flow Cytometry Reagents
Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer

T-Cell Panel

Specificity

Clone

Fluorochrome

PurposeCat. No.

CD3

UCHT1

BD Horizon™ BB515

T cells

564465

CD4

SK3

BD Horizon™ BUV395

Helper T-cell subset

563550CD8SK1

APC-H7

Cytotoxic T-cell subset

560179CD127

HIL-7R-M21

BD Horizon™ BV786

Treg identification

563324

CD25

M-A251

PE

Treg identification

555432

CD27

M-T271

BD Horizon™ BV421

Antigen experience562513

CD45RA

HI100

APC

Antigen experience

550855

CD197/CCR7150503

BD Horizon™ PE-CF594

Antigen experience562381

ProtocolPeripheral blood was stained with antibodies in the presence of BD Horizon™
Brilliant Stain Buffer at room temperature and protected from light for 20 minutes.
Samples were subsequently lysed with BD FACS™ Lysing Solution and washedprior to acquisition.

Data

Analysis of Human T-Cell Populations

104
105

0

CD4+ T Cells

103

104

CD27103104

CD4105

EffectorNaïveCD45RA

104102102

LateCentral
MemoryCentralMemory0Effector
Memory105-1800 102

104105105

Naïve Treg

Treg

0103

CD25104105-2210102

103

CD45RA

104

105

CD127

104-974

103

CD197

Treg

CD4+ T Cells

102

1040103-221

Effector Memory

CD197

105

103

0CentralMemory0 102-102 0 102

CD4+ T Cells-103-1,308-807-221105

Naïve104

CD45RA102-388

Terminal Effector

103-102 0 102-304-221105

CD8+ T Cells-1800105
104

104

103

103

CD27CD8-102 0 102

CD8+ T Cells

103-304-398

103

CD45RA103

CD3+ T Cells-102 0 102

CD45RA

104

105

CD4+ T Cells

105

CD8+ T Cells-304-102 0 102

103

CD197

104

105

Page 4
BD Flow Cytometry Reagents
Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell AnalyzerNaïve, Effector,and Memory
T-Cell Panel

Specificity

Clone

Fluorochrome

PurposeCat. No.

CD3

UCHT1

PerCP-Cy5.5

T cells560835

CD4SK3

BD Horizon™ BUV496

CD4+ subset

564651

CD8SK1APC-H7

CD8+ subset

560179CD27

M-T271

BD Horizon™ BV421

Antigen experience562513

CD28

CD28.2APC

Antigen experience

559770CD45RA

HI100

FITC

Antigen experience

555488CD45RO

UCHL1

BD Horizon™ BUV395

Antigen experience564291

CD197/CCR7150503

BD Horizon™ PE-CF594

Antigen experience562381

ProtocolPeripheral blood was stained with antibodies in the presence of BD Horizon™
Brilliant Stain Buffer at room temperature and protected from light for 20 minutes.
Samples were subsequently lysed with BD FACS™ Lysing Solution and washedprior to acquisition.

DataAnalysis of Human Naïve, Effector, and Memory T-Cell Populations (CD8+)
CD3+ T Cells

105

105

CD8+ T Cells

104
103

CD8103

CD4+ T Cells

-139

00102

103

CD45RA

104

105-1,308-472-102 0 102

CD45RO104

CD8+ T Cells

0-723

103

CD4

104

105

Effector/Effector MemoryNaïve103-360102

CD45RA

104

105

CD8+ T Cells-891

Effector/Effector MemoryCD197

104105Naïve105

105

CD28-346-102 0 102

103

CD28104105-388105-102 0 102-102 0 102
104Naïve103

CD27104104103-388Effector-102 0 102

103

CD27104

CD27103

Central Memory

Early Effector Memory

103-102 0 102
-388

102

Central Memory

105

Late Effector Memory-346

Central Memory0-243-102 0 102

103

CD28104

105

Page 5
BD Flow Cytometry Reagents
Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer

Data

Analysis of Human Naïve, Effector, and Memory T Cell Populations (CD4+)105

CD4+ T Cells

105

CD3+ T Cells

-139

102

103

0-162

102

105

105

104

105

Naïve RTE104103

CD271020 102

103

CD45RA103

CD45RANaïve

104

104
103
102

Effector

000101102

Central Memory
103

CD197

104

-139

Effector Memory-139105

105-102 0 102-388

Central Memory
105105

Effector Memory

Central Memory

104
103

103

CD27104

Effector Memory

CD27

102

Th1 Effector

-388

0

Th1/Th2 Effector/Effector Memory-102 0 102

103

CD28

104

105

-139

CD28

104

102

103

0-102 0 102-139

CD27105NaïveEffector

-139

104

CD4

CD4+ T Cells

105

Effector-388

103-102 0 102

0-304

-388

-102 0 102

CD4+ T Cells

CD45RO103

CD8104104

CD8+ T Cells-388-102 0 102

103

CD28

104

105

103

CD28

104105

Page 6
BD Flow Cytometry ReagentsMulticolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer

Regulatory
T-Cell (Treg)
PanelSpecificity

Clone

Fluorochrome

PurposeCat. No.

CD3

UCHT1

BD Horizon™ BV711

T cells

563725

CD4

SK3

BD Horizon™ BUV395

Helper T-cell subset

563550CD25

M-A251

BD Horizon™ PE-CF594

Treg identification

562403

CD127

HIL-7R-M21

Alexa Fluor® 647

Treg identification

558598

HLA-DR

G46-6

PE-Cy7

Treg subset

560651

CD194/CCR4

1G1

BD Horizon™ BV421

Antigen experience562579CD45RO

UCHL1

BD Horizon™ BB515

Antigen experience564529ProtocolPeripheral blood was stained with antibodies in the presence of BD Horizon™
Brilliant Stain Buffer at room temperature and protected from light for 20 minutes.
Samples were subsequently lysed with BD FACS™ Lysing Solution and washedprior to acquisition.

Data

Analysis of Human Treg Populations

105

105

CD4+ T Cells

104
103

CD127

103

Treg

-1800102

103

104

CD3-221-98

01020 102

CD4

104

CD4+ T Cells

105-98

104

105

105

Early Contact-Dependent Treg

104
102

103

CD45RO

104

Memory Treg

103

CD194103

CD25Naïve Treg

-580102103

CD45RO

104

105-58-15500102102

Naïve and Memory Treg

105Treg0-1800102103

HLA-DR104105

Page 7
BD Flow Cytometry Reagents
Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer

Dendritic Cell
Panel

Specificity

Clone

Fluorochrome

PurposeCat. No.

CD3

SK7

BD Horizon™ BV786

Exclusion (T cell)563799

CD14

M5E2

BD Horizon™ BV786

Exclusion (monocyte)

563698CD19SJ25C1

BD Horizon™ BV786

Exclusion (B cell)563325

CD56

NCAM16.2

BD Horizon™ BV786

Exclusion (granulocyte)

564058

HLA-DRG46.6

BD Horizon™ BV421

Dendritic cells (DC)562804

CD11c

B-ly6

BD Horizon™ BB515

Myeloid DC (mDC)

564490

Clec9a3A4/Clec9A

PE

mDC Subset

563488

CD141

1A4

APC

mDC Subset

564123CD123

7G3

BD Horizon™ BUV395

Plasmacytoid DC (pDC)

564195

ProtocolPeripheral blood was stained with antibodies in the presence of BD Horizon™
Brilliant Stain Buffer at room temperature and protected from light for 20 minutes.
Samples were subsequently lysed with BD FACS™ Lysing Solution and washedprior to acquisition.

DataAnalysis of Human Dendritic Cell Populations103050102100

SSC

HLA-DR

150

104200105250(x1,000)

DC Scatter Gate
Lineage Negative HLA-DR+ DCs

50100150

FSC200250-321

DC Scatter Gate

-398

-102 0 102

105

105
104

CD141

Clec9a– mDC
Clec9a+ mDC

102

103

104
103

0101

-102 0 102

CD123

Plasmacytoid DC
Myeloid DC-388

104

Myeloid DC

105

Lineage Negative HLA-DR+ DCs-139

103

CD3/CD14/CD19/CD56

0102

103

CD11c

104

105-6400103

Clec9A

104105

Page 8BD Flow Cytometry Reagents
Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer

Myeloid CellPanelSpecificityClone

Fluorochrome

PurposeCat. No.

CD3

UCHT1

BD Horizon™ BV510

Exclusion (T cell)563109CD19

SJ25C1

BD Horizon™ BV510

Exclusion (B cell)562947CD20

2H7

BD Horizon™ BV510

Exclusion (B cell)563067

HLA-DR

G46-6

FITC

DC, NK, monocyte subsets

555811

CD163G8

BD Horizon™ BUV737

NK cell/monocyte subset

564434

CD56

NCAM 16.2

BD Horizon™ BV421

NK cells

562751

CD11c

B-Ly6

BD Horizon™ PE-CF594

DC subset

562393CD123

7G3

BD Horizon™ BUV395

pDC subset

564195CD14

M5E2

APCMonocytes

555399ProtocolPeripheral blood was stained with antibodies in the presence of BD Horizon™
Brilliant Stain Buffer at room temperature and protected from light for 20 minutes.
Samples were subsequently lysed with BD FACS™ Lysing Solution and washedprior to acquisition.

Data

Analysis of Myeloid Cells (Dendritic Cells (DC) and Monocytes)
105

250

Dendritic Cells

Granulocytes

Plasmacytoid DC

104

104

200

105

(x1,000)

Granulocyte/DC

Myeloid DC

103
102

Granulocyte/DC0101

50-102 0 102
-472

CD123

SSC

150

Dendritic Cells

100

103

CD14Monocytes-2,789101102103104

CD3/CD19/CD20105

0101102103

HLA-DR

1041050101102103

CD11c104

105

Monocytes

103

Classical0101102

103

HLA-DR104

105

DataAnalysis of Human Natural Killer (NK) Cells

105--2210102

CD16104

105

Non-classicalImmature NK Cells

Mature and Immature NK Cells
105

105

Lineage Neg

Activated NK Cells

0104
102
0102
102

103

104

CD3/CD19/CD20105

-98

0102

103

CD16104

105

Class 1 Laser Product.
Research Use Only. Not for use in diagnostic or therapeutic procedures.
APC-Cy7: US patent 5,714,386
Alexa Fluor® is a registered trademarks of Life Technologies Corporation.
CF™ is a trademark of Biotium, Inc.
Ficoll-Paque is a trademark of GE Healthcare.
Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon
University, and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any otheruse requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.
BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2015 BD
23-17126-00-221

0101-58-263-102 0 102

Lineage Neg

103

CD16104103

CD56103

CD14104

Mature NK Cells

0101

102

103

HLA-DR

104105