BD LSRFortessa™ X-20
15-Color Immunophenotyping

In this experiment, the BD LSRFortessa™ X-20 system was used in combination with BD reagents tostain human peripheral blood mononuclear cells (PBMCs) for analysis of common T-cell, B-cell, NK-cell,dendritic-cell (DC), and monocyte subsets. Three different gating strategies enabled identification of Tcells, NK-T cells, and regulatory T cells (Tregs); B cells, NK cells, and DCs; and monocytes. The five-laser
configuration of the BD LSRFortessa X-20 flow cytometer combined with novel BD Horizon™ Brilliant
Violet™ and BD Horizon™ Brilliant Ultraviolet™ reagents enables optimal panel design based on
antigen density and fluorochrome brightness.
Instrument Configuration
Laser

FilterFluorochrome

Specificity

Clone

Cat. No.

Blue
488 nm

530/30

FITC

CD57

HNK-1

347393

695/40PerCP-Cy™5.5

CD3

SK7340949

Yellow-Green561 nm

586/15PE

CD11c

S-HCL-3347637610/20

BD Horizon™ PE-CF594

CD163G8562293

780/60PE-Cy™7

CD33

P67.6333946

670/30

APC

CD56

NCAM16.2341025

730/45Alexa Fluor® 700

CD202H7560631

780/60APC-H7

HLA-DR

L243

641393450/40

BD Horizon™ BV421

CD123

9F5562517

525/50

BD Horizon™ V500

CD14MφP9562693

610/20

BD Horizon™ BV605

CD25

2A3562660

660/20

BD Horizon™ BV650

CD335 (NKp46)

9E2/Nkp46563230710/50

BD Horizon™ BV711

CD8

RPA-T8563676

780/60

BD Horizon™ BV786

CD19

SJ25C1563325

379/28

BD Horizon™ BUV395

CD4

SK3563550

Red
640 nmViolet405 nmUltraviolet
355 nmProtocolPBMCs were isolated by preparing a mononuclear cell fraction using FicollPaque™ Plus. Briefly, 15 mL of whole blood was diluted with 15 mL of phosphatebuffered saline (PBS) + 2% fetal bovine serum (FBS). The entire 30 mL of diluted
blood was layered over 15 mL of Ficoll-Paque Plus in a 50-mL Falcon® tube.
Blood was centrifuged at 400g for 30 minutes at room temperature with thebrake off. PBMCs were removed from the plasma-Ficoll interface and washedtwice with BD Pharmingen™ stain buffer. Cells were counted and aliquoted
at 1 x 106 cells per tube and incubated with antibodies on ice for 20 minutes,
washed, and acquired on a BD LSRFortessa X-20 flow cytometer. Single cells
were identified by gating on FSC-A vs FSC-H. Lymphocytes or monocytes werethen identified based on FSC vs SSC and further analyzed as described in thesubsequent figures.

BD LSRFortessa™ X-2015-Color Immunophenotyping

Page 2Data

Distinguishing helper and cytotoxic T cells, Tregs, and NK-T cells

100

Count

150

200

250

A

CD3–050

T Cells

0103

104

105

CD3 PerCP-Cy5.5

E201510Count104
103

CD56+ NKT Cells

0CD8 BV711

CD8+ T Cells

T Cells

25T Cells
105

B50-103

CD4+ T Cells

0102103104105-103

0CD4 BUV395

CD57 FITC

104103105

CD56+ NKT Cells
CD57+ CD8 NKT

CD57+ CD8 Int NKT

CD8– CD57– NKT

CD8+ NKT

CD8 Hi CD57+ NKT

00-103

CD25 BV605

105

104

F

Regulatory T Cells

0101023104105CD4 BUV395-1030103

CD8 Hi CD57– NKT

104105CD8 BV711

Regulatory T Cells
105

D104103

CD4+ T Cells
105C103

CD56 APC

104
1030-103

CD25 BV605

Activated Tregs

0103

104105

HLA-DR APC-H7Discussion

The CD3+ population (A) contains traditional CD4 and CD8 T cells in addition toTregs and natural killer T cells. Gating on CD4+ T cells (B) allows the identificationof CD25+ Tregs (C) that can be further analyzed for activation based on HLA-DRexpression (D). Natural killer T cells are characterized by CD56 expression within
the CD3+ subset (E). With the use of multicolor flow cytometry, we can identify
NK-T–cell subsets based on CD57 and CD8 expression (F).

BD LSRFortessa™ X-2015-Color ImmunophenotypingA250

BCD20 Alexa Fluor® 700CD3–T Cells

-104

0104

104

105

CD3–0103

B Cells
Non-B/T/NKT Cells

-103

50100

150

Distinguishing B cells, NK cells, and DC subsets

SSC

Data200

Page 3-103

105

0

CD3 PerCP-Cy5.5

CD57– CD56+ NK Cells

104

NKP46+ CD16–

NKP46– CD16+

CD57– CD56+ NK Cells
103

104

-102 0 102

105

CD56 APC

104105

105
104

Plasmacytoid

103

CD123 BV421

Dendritic Cells

0 102

1,500
500

Count

2,000

F

Dendritic
Cells

1,000

103

CD16 PE-CF594

Non-B/T/NKT/NK Cells

E

NKP46+ CD16+103

103

Non-B/T/NKT/NK Cells

0105

105

DCD335 (NKp46) BV650

104

105

CD57+ CD56+ NK Cells

-103

104

0

Non-B/T/NKT Cells

0CD57 FITCC103

CD19 BV7860Myeloid0103

104

HLA-DR APC-H7Discussion

105

0103

104

105

CD11c PEGating CD3– negative cells (A) allows for identification of B cells, NK cells, andDCs. CD19 and CD20 identify the B-cell population, while cells negative for CD19/
CD20/CD3 (B) include NK cells and DCs (C). CD56 and CD57 identify functionallydistinct mature NK cell populations from other subsets of NK cells based on CD16and CD335 expression (D). Remaining PBMCs contain dendritic cells that can be
identified as CD123hiCD11c– plasmacytoid and CD123–CD11chi myeloid subsets(E, F).

BD LSRFortessa™ X-2015-Color Immunophenotyping
Data
250

Identification of pro-inflammatory monocytes

150
100

SSC

200

A

Page 450

CD3– Cells

0103

104

105

CD3 PerCP-Cy5.5

105
104

CD19 BV786

CD3– Cells

Non-B/T/NKT/NK Cells-103 0 103

B-103

0103

104

105

CD56 APC

HLA-DR APC-H7

104
103

105
00 102

CD14 V500

Monocytes

Monocytes
Pro-Inflammatory Monocytes

104D103

Non-B/T/NKT/NK Cells105C-1030103

104

CD33 PE-Cy7105-102 0 102

103104

105

CD16 PE-CF594

Discussion

Monocyte gates based on light scatter can be further refined by gating out T, B,and NK cells based on CD3, CD19, and CD56 respectively (A, B). Monocytes areclearly identified based on CD14 and CD33 expression (C). Pro-inflammatory
monocytes are identified as CD16hiHLA-DRhi cells (D).

Conclusion

A five-laser BD LSRFortessa X-20 system and BD flow cytometry reagents enablehigh-order multiplexing of results. In this 15-color example, we were able to subset
PBMCs into 7 unique populations.

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For Research Use Only. Not for use in diagnostic and therapeutic procedures.
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CF is a trademark of Biotium, Inc.
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