BD FACSymphony™ A5 SE
Cell Analyzer
Unlock the full potential of the BD FACSymphony™ platform withthe flexibility of spectral unmixing or compensation workflows

Five lasers and 48 detectors for maximum
coverage of the fluorochrome emission spectrum
Access spectral abilities

Choose between live spectral orcompensation-based workflows inon your BD FACSymphony™
A5 System with aBD FACSymphony™ A5 SE
System upgrade kit

BD FACSDiva™ Software

Resolve criticalcell populations

Perform spectral unmixing live onBD FACSDiva™ Softwarewith high autofluorescence
using autofluorescence unmixingor export the data for analysis in FlowJo™
Software (v10.6 or later)

The BD FACSymphony™ A5 SE Cell Analyzer enables bothspectral unmixing and compensation workflows• Algorithmically optimized filter set collects
the full spectrum of emitted light• Algorithm trained on available
fluorochromes and scalable detector array
technology allows optimized placementof 48 detectors across five on-board lasers• High-performance PMTs forenhanced resolution

BD FACSymphony™ A5Cell Analyzer

BD FACSymphony™ A5 SECell Analyzer• Using the familiar BD FACSDiva™
Software workflow, assign afluorochrome to a detector
with the option to analyze
using compensation or spectral
unmixing live during acquisition• Visualize fluorochrome profiles
with spectral plots

Figure 1. Comparison of common BD FACSymphony™ A5 and A5 SE Cell Analyzer
filter configurations
2• Extract autofluorescencefrom highly fluorescentcells to potentially improvepanel resolution

Distinguish between spectrally similar fluorochromes
The BD FACSymphony™ A5 SE Cell Analyzer features an optimized filter set to collect the full spectrum of emitted light, increasing
flexibility in fluorochrome choices and enabling simultaneous analysis of fluorochromes with similar spectral signatures. The
following panel contains a total of 40 fluorescence reagents. It includes the new spectrally optimized fluorochromes, BD Horizon
RealYellow™ and RealBlue™ Reagents and was used to characterize a variety of immune cell populations in human peripheral blood.
CD45+

200K

100K

200K

150K

SSC-A

SSC-A

150K

250K

Live
cells

100K

150K
100K

50K

50K

50K

000050K 100K 150K 200K250K-10FSC-A

4010410FVS440UV5-1030CD123−CD303−

510CD45+cellsCD303 BV711

Singlets

200K

FSC-H250K

10310410CD45 NFB530

5101410231003-105-104010410CD19 PE-Cy5.5Live cells
250K

1. pDC
2. Basophils
3. B cells
4. T cells
5. CD19−CD3−

404-105341050CD123 Spark NIR 685

10410CD3 BV570

5Monocytes

1041050K

0050-10010105-1041060
40204105-10-10

30103104

Monocyterelated DC010CD11c BB630-1040104CD16 BV510

10104105-1041005-1040104105105400104104HLA-DR BUV496

105HLA-DRhighCCR2low

1051041004-105Intermediate
monocytes-1040104105CD14 Spark YG 593

0104105KIR+ NK-104010410CD158 RY586

80604041031003-1020050104CX3CR1 BUV615

5101031003-10-1054103010310IgD PE

4105-100103104CD11c BB630

10310410IgM BV750

31003-105010410CD1c BV65050104CX3CR1 BUV615

100104CD3 BV570

105031003-10Plasmablasts

50104105CD38 BUV737• Plasmablasts • Mature B• Memory B • Immature B

104101031011050104105-1030CD38 BUV737

510410123101303-100104CD161 BUV661

14. Central memory CD4 T cells
CD4 T cells

10CD8T cells

410CD4T cells

050104CD4 R718

CD45RA+CCR7+510CCR7 BV421

TCRɣб−3-10510CD8 PerCP

CD45RA BUV395ɣб T cells

410410CD38 BUV737

CD3+TCRɣб−

CD3+TCRɣб+3103100IgD−CD27+
Memory B cells

103104IgM BV750

105CD4 T cells

T cells
5104105510IgD+CD27−
CD24 APC-H741012. CD161low ɣб T cells 13. CD161high ɣб T cells

01051010510CD27 PE-Cy731003-10

CD24 APC-H7FcεR1α BB700

4104100B cells
510Myeloid cDC251033

10. Immature B cells
11. Mature B cells

510-10IgM Memory B cells

41031003-10145010410410CD45RA−CCR7−510Naïve
CD4T cells

CD27 PE-Cy740IgD+CD27+

CD20 Amethyst Orange

9310B cells• Monocyte-related DC • Non-classical monocytes • Myeloid cDC2 • pDC

31003-105-10CD45RA BUV395

30103104

CD28 BB515

105410310EffectormemoryCD4T cells

03-10-1030103104

CD28 BB515

105Regulatory T cells

CD8 T cells• Central memory CD8 T• Effector memory CD8 T• Naive CD8 T

16. Central memory CD8 T cells 17. Effector memory CD8 T cells
18. RA effector memory CD8 T cells
CD45RA+CCR7+

CD8 T cells

CCR7 BV421

170104181041031003-105NaïveCD8T cells-10CD45RA BUV395

301051041031003-10310410CD95 BV786

5-1030103104TIGIT BB660

10-10530103104CD25 PE-AF700

105510Naïve Treg
1541031003-1000104HLA-DR BUV496

1052031003-100104CD161 BUV661

10541031003-100104CD279 RB780

105CX3CR1 BUV615

31003-10410CD27 PE-Cy74104105-1030103104TIGIT BB660

105CD28−CD27−
510510CD28 BB515

CD45RO BUV563

1951010CD39 PE-Fire810

Memory CD8 T cells (CD8+minus Naïve)510• CD28+CD27−• CD28+CD27+

CD127low CD25high

CD4 T cells
CD127 BUV805

TSCM5101641015. Memory Treg

CD45RA BUV395

HLA-DR BUV496

5CD14 Spark YG 593

Dendritic cellsTCRγδ PE-Cy5100-10CD62L BV480

0410CD56 VioBright R667CCR7 BV421

4Classicalmonocytes

4-10510100

CD27 PE-Cy78Normalized To Mode

CD57 VioBlue

410-10CD38 BUV737

10410CD14 Spark YG 593• Immature NK• Mature NK

51075104-1031003-100CD64 APC

0B cells
7. Mature NK 8. Immature NK 9. CD56−CD16−
Lymphocytes and DC
Mature NK

5104410

HLA-DRlowCCR2high

65104-10NK cells

CD16 BV510

00

CD14 Spark YG 593

410544106. Non-classical monocytes
CD64low

Lymphocytes & DC0CD45RO BUV563

10SSClowCD14−
51080

5004-10CD14 Spark YG 593

CD86 BB755

31003-10410100

Normalized To ModeFcεR1α BB700

Basophils

41034• Basophils • pDC

CD64−CD45RA−

310HLA-DR BUV496

SSClow
CD14−

410CCR2 BV605

20CD45RA BUV395

510100K

CD86 BB755

150K

40

CD16 BV510

0200K

60

CD64high
510510510CD16 BV510

31003-1080

SSChighCD14+

CD16 BV510

pDC

SSC

Normalized To ModeFcεR1α BB700

410250K

SSChighCD14+CD56−

SSChighCD14+CD56−

CD19−CD3−

100

CD16 BV510• Basophils • pDCpDC
51041031003-1021-1030103104CD28 BB515

10551019. Chronically activatedCD8 T cells
20. CD161+ memory
CD8 T cells
21. Terminally differentiatedCD8 T cells

41031003-100103104CD57 VioBlue

1053Gating strategy for identification of immune cell subsets in human peripheral blood using a 40-color panel
Cell populations

Surface phenotype

Live leukocytes

Singlets+FVS440UV-CD45+

Basophils

Singlets+FVS440UV-CD45+CD123+CD303-CD64-CD45RA-FcεR1α+CD45RO+Plasmacytoid dendritic cells (pDC)

Singlets+FVS440UV-CD45+CD123+CD303+CD45RA+FcεR1αlow

Monocytes

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSChighCD14high/lowCD56-

Monocyte subsets
Dendritic cells

Classical monocytes

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSChighCD14high/lowCD56-CD86high/lowCD64highCCR2highHLA-DRlowCD14highCD16low

Intermediate monocytes

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSChighCD14high/lowCD86high/lowCD64highCCR2lowHLA-DRhighCD14highCD16+

Nonclassical monocytes

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSChighCD14high/lowCD86high/lowCD64lowCD14lowCD16high

Monocyte-related DC

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSClowCD14-CD86+CD16+

Myeloid dendritic cells (cDC2)

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSClowCD14-CD86-CD16-CD56-HLADR+CD11c+CD1c+FcεR1α+CX3CR1low

NK cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSClowCD14-CD86-CD16+CD56high/low

NK cell subsets

Immature NK cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSClowCD14-CD86-CD16+CD56highCX3CR1-

Mature NK cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSClowCD14-CD86-CD16+CD56lowCX3CR1+KIR+CD57+ NK cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19-SSClowCD14-CD86-CD16+CD56lowCD158+CD57+

B cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19+

B cell subsets

Immature B cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19+CD27-IgD+CD24highCD38highIgMhigh

Mature B cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19+CD27-IgD+CD24+CD38+IgMlow

IgM memory B cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19+CD27+IgD+CD62L+IgMhigh

Memory B cellsSinglets+FVS440UV-CD45+CD123-CD303-CD3-CD19+CD27+IgD-IgM-

Plasmablasts

Singlets+FVS440UV-CD45+CD123-CD303-CD3-CD19+CD27+IgD-CD20-CD38highIgM-

T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-γδ T cellsγδ T cell subsets

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ+CD161high γδ T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ+CD45RA+CD161high

CD161low γδ T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ+CD45RA+CD161low

CD4 T cells
CD4 T cell subsets

T cell subsets

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8-CD4+Naïve CD4 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8-CD4+CD45RA+CCR7+

Central memory CD4 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8-CD4+CD45RA-CCR7+

Effector memory CD4 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8-CD4+CD45RA-CCR7-CD28+CD27+TIGIT+/-

Effector Th1 CD4 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8-CD4+CD45RA-CCR7-CD28+CD27-TIGIT-Naïve regulatory T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8-CD4+CD127lowCD25highCD39-CD45RA+

Memory regulatory T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8-CD4+CD127lowCD25highCD39+CD45RA-

CD8 T cells
CD8 T cell subsets

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-Naïve CD8 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-CD45RA+CCR7+CD95-

T stem cell memory cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-CD45RA+CCR7+CD95+

Central memory CD8 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-CD45RA-CCR7+TIGIT+/-

Effector memory CD8 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-CD45RA-CCR7-

RA effector memory CD8 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-CD45RA+CCR7-TIGIT+/-

Terminally differentiated CD8 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-[CD45RA+CCR7+]-CD28-CD27-CX3CR1+CD57+

Chronically activated CD8 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-[CD45RA+CCR7+]-HLA-DR+CD38+

CD161+ memory CD8 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-[CD45RA+CCR7+]-CD161+

CD279-expressing CD8 T cells

Singlets+FVS440UV-CD45+CD123-CD303-CD3+CD19-TCRγδ-CD8+CD4-[CD45RA+CCR7+]-CD279+

This gating strategy allows identification of a selected number of cell populations after staining of human peripheral blood mononuclear cells (PBMCs) witha cocktail of 39 antibodies and a viability dye.

Class 1 Laser Product.
For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD Horizon RealBlue™
Reagents are not released and will be available in the Fall 2022

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