BD LSRFortessa™ X-20 System
5-Color Panels Designed for Minimal Compensation

In this experiment, a five-laser BD LSRFortessa™ X-20 flow cytometer was used in combination
with BD reagents to design panels that are optimized for minimal compensation and optimal signal
by selecting one bright fluorochrome per laser. Three different panels are shown: human T cell,
human B cell, and mouse B cell.
Analyzer Configuration
Laser

FilterFluorochrome

Human T-Cell PanelBlue 488 nm

530/30FITC

CD8IgD

IgD

Yellow-Green 561 nm

610/20PE-CF594

CD27

CD38IgM

Red 640 nm

670/30APC

CD45RA

IgM

CD21

Violet 405 nm

450/40

BV421

CD3

CD27

CD23

Ultraviolet 355 nm

379/28

BUV395

CD4

CD19

CD19

Compensation*
Fluorochrome(-)% Fluorochrome

FITC
PE-CF594
APC% Compensation
0.00%

BV421

0.00%
0.00%

BUV395

0.00%

BV421

0.02%

PE-CF594
APC

FITC

0.00%
0.04%

BUV395

0.00%

BV421

0.00%

FITC
APC

PE-CF594

0.29%
1.39%

BUV395

0.00%

BV421

0.00%

FITC
PE-CF594

APC

0.00%
0.03%

BUV395

0.00%

BV421

1.57%

FITC
PE-CF594
APC

BUV395

0.00%
0.00%
0.24%

*Representative compensation values. Compensation varies as a function of PMT voltage.

Human B-Cell Panel

Mouse B-Cell PanelBD LSRFortessa™ X-20 System
5-Color Panels Designed for Minimal Compensation
ProtocolPage 2

PBMCs were prepared by using Ficoll-Paque™ Plus according to themanufacturer’s directions and incubated with antibodies at room temperature
protected from light 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 were then identified based on FSC vs SSC and further analyzed as
described in the subsequent figures.
For mouse experiments, C57BL6 spleen was harvested into a single-cellsuspension using a cell strainer and syringe plunger. Red blood cells were lysedusing BD Pharm Lyse™ lysing buffer (Cat. No. 555899) on ice for 2 minutes.
Cells were subsequently washed, stained with antibodies for 30 minutes on ice,
washed, and acquired on a BD LSRFortessa X-20 flow cytometer.

DataAnalysis of Human Naïve, Effector, and Memory T Cells

CD8+103

CD4+
0 102

1,000

CD8 FITC

1,500

104

CD3+0500

Count2,000

105

2,500

CD3+0102103

1041050102

CD3 BV421

1041051041030 1020 102103CD45RA APC

10410105

CD3+/CD4+

5CD3+/CD8+CD45RA APC103

CD4 BUV395

0102103

104

CD27 PE-CF594

Discussion

105

0102

103

104

105

CD27 PE-CF594
Human PBMCs were stained with CD4 BD Horizon™ Brilliant Ultraviolet™ 395(BUV395), CD8 FITC, CD3 BD Horizon™ Brilliant Violet™ 421 (BV421), CD27PE-CF594, and CD45RA APC. Singlet lymphocytes were discriminated basedon light scatter properties, and the CD3+ population was identified (top left).
Traditional CD4 and CD8 T-cell subsets can be gated on from the CD3+ lymphocyte
population (top right). Within the CD8+ (bottom left) and CD4+ (bottom right)
T-cell subsets, naïve (CD27+CD45RA+), memory (CD27+CD45RA–), and effector(CD45+/–CD27-) T cells can be identified.

BD LSRFortessa™ X-20 System
5-Color Panels Designed for Minimal Compensation
Data

Analysis of Human B-Cell SubsetsNaïveNon-Switched Memory

Double Neg

Switched Memory

5CD19+00 102103

IgD FITC104

7.5105

10CD19+2.5Count

Page 30102103104

1050102

CD19 BUV395
Naïve105

105

0 100 10210103

IgM APC

10104

105432IgM APC

104

Non-Switched Memory
Transitional

0102

103

104

105

0102

CD38 PE-CF594

103

104

105

CD38 PE-CF594

104
103
0 102

0 10

2103Plasmablasts

IgM APC

10410105

Double Negative5

Switched Memory

IgM APC103

CD27 BV421

0102

103

104

CD38 PE-CF594

Discussion

1050102

103

104

105

CD38 PE-CF594
Human PBMCs were stained with CD19 BUV395, CD27 BV421, IgD FITC, CD38
PE-CF594, and IgM APC. Singlet lymphocytes were discriminated based on lightscatter properties, and the CD19+ population was identified (top left). Naïve, nonswitched, switched memory, and double negative B-cell subsets can be identified
from the CD19+ lymphocyte population based on expression of CD27 and IgD(top right). Within all subsets, IgM vs CD38 expression was analyzed (middleand bottom). Within the naïve B-cell subset (middle left), transitional B cells canbe identified as IgM++CD38++. Within the switched memory subset (bottom left),plasmablasts can be identified as IgM–CD38++.

BD LSRFortessa™ X-20 System
5-Color Panels Designed for Minimal Compensation
Data

104

105

104

102103104

105105102103

104105105103

104105104105

IgD FITC

CD19 BUV3950 102
0-245

102

103

104

IgM PE-CF594

105-132

104

102

105

105
104

CD23 BV421

0 102
103

IgM PE-CF594

-119

102

0-95

IgD FITC

103

105
104
103
0 102
0-245

104

CD19 BUV395

0 102
0-95-132

104

104

103

IgD FITC

103

1051040 102
102-1140-95

105CD23 BV421

103

CD21 APC

104

1050-28103

103

CD19 BUV395

103

IgM PE-CF594

103

CD21 APC

1051020 102-245

Follicular-140CD21 APC

Marginal Zone

0 102

CD19+0Count100 200 300 400 500 600 700

Analysis of Mouse Marginal Zone and Follicular B Cells-132-114

Page 40-245

102103IgM PE-CF594

Discussion

C57BL6 splenocytes were stained with CD19 BUV395, CD23 BV421, IgD FITC,IgM PE-CF594, and CD21 APC. CD19+ B cells (top left) can be subdivided into
follicular and marginal zone cells based on expression of CD23 and CD21 (topright). The variable expression of CD21, CD23, IgM, and IgD is shown for thetwo subsets in the middle and bottom rows. The marginal zone cells show higher
expression of IgM and CD21 compared to the follicular cells. In comparison, theexpression of CD23 and IgD is higher on the follicular cells.

Conclusion

The five-laser configuration of the BD LSRFortessa X-20 system, combinedwith novel BD Horizon™ Brilliant Violet™ and BD Horizon™ Brilliant
Ultraviolet™ reagents, enables optimal panel design based on antigen densityand fluorochrome brightness while optimizing signal by using all five lasers
simultaneously.

Class 1 Laser Product.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
CF is a trademark of Biotium, Inc.
Ficoll-Paque is a trademark of GE Healthcare.
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23-15823-00