BD LSRFortessa™

Performance without peer,
choice without compromisePerformance without peer,
choice without compromise
The BD LSRFortessa™ cell analyzer offers the ultimate in choice for flow
cytometry, providing power, performance, and consistency. Designed tobe affordable and expandable, the BD LSRFortessa has the flexibility tosupport the expanding needs of multicolor flow cytometry assays.
The BD LSRFortessa analyzer can be configured with up to 4
lasers—blue, red, violet, and UV—which enables the detection of up
to 18 colors simultaneously. The BD LSRFortessa may be upgraded
subsequently with additional or new lasers from BD, as future
requirements dictate.
Innovative Designs for Optical Efficiency
Built on a proven platform, BD LSR analyzers
feature innovative designs for both theexcitation optics and collection optics that
reduce excitation losses and improve lightcollection efficiency. The result is optical
efficiency that delivers maximum sensitivity
and resolution for multicolor applications
by reducing excitation laser light loss, andimproved fluorescent signal collection.

Special Order Program
For research needs at the leading edgeof biomedical discovery, the special orderprogram offers BD LSRFortessa researchers
the latest innovations in laser technology
to enable future upgrades with new
excitation, wavelength, and power choices.
The program lets customers fully customize
configurations to deliver added flexibility
and power to support advanced research.
Technical and Application Support
As with all BD instruments and reagents,
highly qualified BD technical and application
support personnel are available for help
in streamlining research and maintaining
optimal instrument performance.

Boundless Potential

The BD LSRFortessa Analyzer
Flexibility in a Small SpaceAn affordable choice to fit most flow cytometry analyzer
needs, the BD LSRFortessa can be ordered with up to 4
lasers—blue, red, violet, and UV—which provides flexibility
in laser wavelengths so assay design can be optimized using
the latest fluorescent dyes and substrates. The instrument
can accommodate the detection of up to 18 colors
simultaneously with a defined set of optical filters thatmeet or exceed the majority of today’s assay requirements.

The BD LSRFortessa is designed to enable your current assay
requirements. As future needs arise, lasers can be added or
upgraded.

The BD LSRFortessa puts the power of the proven BD LSR
platform into a compact footprint. It can easily fit on thebenchtop for cost-effective space use. This space efficiency
is even more important for labs with limited space or where
space cost is premium.
In addition to the reduced size, design enhancements
provide easier access to bandpass filters and mirrors,
simplifying changes to experimental setup.
To improve experimental workflow, the optional
BD™ High Throughput Sampler (HTS) provides rapid, fully
automated sample acquisition from microtiter plates. In
high-throughput mode, the HTS option can speed througha 96-well plate in less than 15 minutes with less than 0.5%
sample carryover from one well to the next. Low carryoveris essential in research applications to ensure sample purity
and data integrity.

BD High Throughput Sampler (HTS)

Analysis of Regulatory T Cells

4

All Events

Lymphocytes
105

3,500 4,000

CD3+

102

103

104

105

104
103

1,000
500
0

0

CD8 PerCP-Cy5.5

2,500

CD3+

1,500 2,000

Count

3,000

(x 1,000)
250
200

CD4+CD8neg
102

CD3 APC-H70

Lymphocytes
50

100

150

FSC

200

250
(x 1,000)

-132

SSC

150
100

-140

50

Regulatory T cells (Tregs) play a critical rolein maintaining immune modulation and arepresent in normal peripheral blood in low
numbers (5 to 10% of CD4+ T cells or 1 to
2% of total lymphocytes). Forkhead box P3(FoxP3) is considered to be the definitivemarker for this rare cell population.
Tregs are known to express the highest
levels of CD25. The CD4+CD25+(high) gating
strategy shown here was used to identify
Treg populations. These plots represent
data taken from a BD LSRFortessa using
an 8-color panel that applies this gating
strategy to identify FoxP3-positive cells.

0 102

102

103

104

CD4 BD Horizon V500

105

BD LSRFORTESSA

CD25+CD127lo

CD4+CD8neg

CD25+CD127lo

CD4+CD8neg-471-102 0 102

103

CD25 APC

104

105

104
103

IFNγ+CD4+#Events %Parent %Total
960,606####
100.0
238,14424.8
24.8
108,466
45.5
11.3
63,347
58.4
6.6
4,059
6.4
0.4
3,192
78.6
0.3
3,360
82.8
0.3
699
17.2
0.1
1,921
3.0
0.2

102

-92

101

0

0

102

103

104

FoxP3 Alexa Fluor® 488

105

0

-471

0

-194

-102

-102

0

10

102

CD25+CD127lo

Population
All Events
Lymphocytes
CD3+
CD4+CD8neg
CD25+CD127lo
FoxP3+
CD45RA+CD25+
CD45RAnegCD25+
IFNg+CD4+102

CD4 BD Horizon V500

103

CD25 APC

40
20

30

Count

104
103

CD127 PE

CD45RA+CD25+

104

50

CD45RAnegCD25+

FoxP3+

Stimulated

105

105

60

105

Tube:

-313

-102 0 102

103

104

CD45RA BD Horizon V450

105

-265

-102 0 102

103

104

105

IFNγ PE-Cy7

5

FLUIDICS

Superior Performance

Unique and Revolutionary Designs
for Multicolor Analysis
Fluidics System
The fluidics system is pressure driven. Hydrodynamic focusing
forces sample cells through the cuvette flow cell, where
they are interrogated. The flow cell is in fixed alignment
with the laser and gel-coupled to the collection optics.
This design ensures that the laser is precisely focused on
the sample stream and the maximum amount of emitted
light can be collected for added sensitivity in multicolor
applications. Fixed alignment also minimizes startup time,
improves experiment-to-experiment reproducibility, andenables automated daily quality control.

Many innovations are incorporated into the BD LSRFortessa
product line. The heart of the cytometer, the fluidics system
features a true fixed-alignment flow cell that is gel-coupled
to the collection optics to maximize detector signal.

The sheath container (8 L) and waste container (10 L) areoutside the cytometer, and positioned on the floor foreasier access.
The optional BD FACSFlow™ supply system fluidics cart
increases capacity and ease of use while maintaining a stablefluidics pressure. It includes an automated sheath and waste
fluid control system that reduces daily maintenance byincorporating two 20-L containers (Cubitainers®).
Fluidic sensors maintain constant pressure, while a fluidics
monitoring system warns when sheath fluid is low or empty,
or when the waste container is full.
Figure A-1 Hydrodynamic focusing of the sample core through the flow cell

Low Sample
Pressure(12 µL/min)

Laser BeamHigh Sample
Pressure(60 µL/min)

Laser Beam

Figure A-1 Hydrodynamic focusing of the sample core through the flow cell

Low Sample
Pressure(12 µL/min)

Laser Beam

SheathFluid

SheathFluid

6

SampleSampleSheathFluid

SheathFluid

High Sample
Pressure(60 µL/min)

Laser Beam

SheathFluid

SheathFluid

SampleSampleSheathFluid

SheathFluid

Hydrodynamic focusing of the sample
core through the flow cellCuvette flow cell

CuvetteLaser Beams
Cuvette flow cell

Flow Cell

A quartz cuvette flow cell is gel-coupled
to the collection optics. This design helps
ensure that lasers are precisely focused on
the sample stream to generate the greatest
signal and ensure that the maximum
amount of emitted light is collected.

Sample Injection Tube (SIT)

7

OPTICS

Maximum Signal, Minimum Crosstalk

An Innovative and Proven Platform
for Multicolor Analysis
Optics System
The optics system consists of laser excitation optics that
illuminate cells in the sample, and collection optics that
direct light scatter and fluorescence signals through spectral
filters to detectors. Innovative designs for both the excitation
optics and collection optics in BD LSRFortessa systems reduce
excitation losses and optimize collection efficiency forincreased sensitivity and resolution.

The patented collection optics are yet another design
innovation. Arranged in octagon- and trigon-shaped opticalpathways, their novel design efficiently maximizes signal
detection and increases sensitivity and resolution. This
allows researchers to identify cells, especially dim and rarecell populations, optimizing multicolor assays and panel
design for superior results.

Excitation Optics
The excitation optics consist of multiple fixed wavelength
lasers, beam shaping optics, and individual pinholes which
result in spatially separated beam spots.
A final lens focuses the laser light into the gel-coupledcuvette flow cell. Since the optical pathway and the sample
core stream are fixed, alignment is constant from day to day
and from experiment to experiment.

Normalized Emission (%)

Emission spectra of commonly used fluorochromes
PerCP

100
PE

AmCyan80

Alexa Fluor® 700

BD Horizon™ V450
FITC
APC

60

APC-H7PerCP Cy™5.5

40
PE-Cy™7

20
0
400

8

500

600

700

Wavelength (nm)800

Collection Optics
Emitted light from the gel-coupled cuvette is deliveredby fiber optics to the detector arrays. The collection optics
are set up in patented octagon- and trigon-shaped opticalpathways that maximize signal detection resulting fromeach laser illuminated beam spot. Bandpass filters in frontof each PMT allow spectral selection of the collected
wavelengths. Importantly, this arrangement allows filter
and mirror changes within the optical array to be madeeasily and requires no additional alignment for maximum
signal strength.

This design is based on the principle that light reflection ismore efficient than light transmission. Emitted light travels toeach PMT detector via reflection and is transmitted throughonly two pieces of glass to reach the detector. Therefore,
more colors can be detected with minimum light loss.

D

B

F

Transmission pathways in an octagon

H
G

A
E
C
9

Greater Consistency

Automated Controls for Setup,
Acquisition, and Analysis
Cytometer Setup and Tracking
The Cytometer Setup and Tracking (CS&T) feature of
BD FACSDiva software establishes baseline settings andadjusts for instrument variability. The software reduces
operator error, and ensures consistency of results by setting
the signal time delay across the multiple laser beams andoptimizing PMT voltages. Application-specific settings canalso be set, allowing for rapid setup and performanceof routine experiments in a more consistent manner.
Quality control (QC) tracking capabilities in the software
measure instrument settings and report on performance.
Levey-Jennings plots help users understand instrument
performance and identify maintenance issues.

BD FACSDiva™ software controls the efficient setup,acquisition, and analysis of flow cytometry data fromthe BD LSRFortessa workstation. The software is common
across BD FACS™ instrument platforms, including the
BD FACSCanto™ cell analyzer and BD FACSAria™ cell
sorter systems. This affords researchers greater application
flexibility, allowing them to easily move assays from one
platform to another.

Analysis of human blood
Data shows a 10-color panel run on a 3-laser BD LSRFortessa witha blue/red/violet configuration. The panel was used for defining
T-cell subsets using lysed whole blood (BD Pharm Lyse™ lysing
buffer) from a normal donor. Use of the BD Horizon™ V450 and
BD Horizon V500 dyes allows brighter fluorochromes to be used formore dimly expressed antigens, while the use of 10 colors expands
the amount of information gained from a single tube.

10

105

0 102

103

TCR αβ FITC

104

105

105
104
103

CD8 PerCP-Cy5.5

104
0

0

105

CD8+ T Lymphs

103

CD8 PerCP-Cy5.5

104
103
102
-466

T Lymphs

0

103

104

CD3 Alexa Fluor® 700

105

-466

0

103

104

CD3 Alexa Fluor® 700

105

-108

0 102

103

104

CD4 BD Horizon V500

105

-45

104

T Lymphs

-693

103

CD45 APC-H7

CD4+ T Lymphs

-693

102

T Lymphs

0

150

SSC

100
50
101

-108

CD4 BD Horizon V500

105

250

T Lymphs

200(x1,000)

T Lymphs

-108

0

103

104

CD3 Alexa Fluor® 700

105

BD FACSDIVA

Acquisition and Analysis
BD FACSDiva software enables researchers to preview
and record data from multiple samples with an automatedacquisition process. Acquisition templates, user-defined
experiment layouts, and simple compensation procedures
are also managed by the software to further facilitate
acquisition.

LSRFortessa - T Cells

104
103

150

CD2 FITC-A

Grans

100

SSC-A

200

105

250

LSRFortessa - T Cells

Lymphs
102

103

104

-369

50

0 102

Monos

105

0
-597

CD45 V500-A

For analysis, the software includes powerful features suchas hierarchical snap-to gating, a variety of plot formats, andbatch analysis. Recorded data can be analyzed by creatingplots, gates, population hierarchies, and statistics views on a
BD FACSDiva global worksheet. Once the global worksheet
is saved, it can be used to analyze multiple sample tubes
from an experiment, thereby saving time. Numerous otherproductivity benefits include user-defined batch analysis
and automated features such as gate resizing, pausing
between data files, exporting statistics, and printing before
proceeding to the next data file.

Tube: T Cells

104

T Helper

103

Population
All Events
LymphsT Cytotoxic
T Helper

0

CD4 Alexa Fluor® 700-A

105
104
103

Monos

-722

-351

0

CD8 PE-A

105

LSRFortessa - T Cells

T Cytotoxic

0
-597

103

104

105

0
-597

CD3 APC-H7-A

103

104

105

Grans

CD3 APC-H7-A

104
103
0

-495

-1,435

0

103

104

CD16 PE-Cy7-A

105

LSRFortessa - T Cells

105

LSRFortessa - T Cells

CD56 APC-A

104

105

LSRFortessa - T Cells

103

CD3 APC-H7-A

0
-597

103

104

105

0
-597

CD3 APC-H7-A

103

104

105

CD3 APC-H7-A

BD FACSDiva worksheet showing well defined T-cell population
subsets acquired using the special order BD LSRFortessa analyzer
configured with the following lasers: 60-mW 355-nm UV, 100-mW
450-nm violet, 100-mW 488-nm blue, and 40-mW 640-nm red.

104

105

Tube:

T Lymphs

103

104

Population
All Events
Lymphocytes
CD4+ T Lymphs
CD8+ T Lymphs

0

0

103

CD25 PE-Cy7

104

105

0 102

103

104

CD8 PerCP-Cy5.5

105

T Lymphs

-108

0 102

103

104

CD4 BD Horizon V500

105

-293

0

103

104

CD2 PE-Texas Red®

105

-472

103

-693

0

CD3 Alexa Fluor® 700-129

CD62L BD Horizon V450

105
104
103

T-Cell γδ APC

0-1,098-466

T Lymphs
105

T Lymphs

T Lymphs

-466

0

103

104

105

CD3 Alexa Fluor® 700

11

CUSTOM

Choices Now and in the Future

Special Order Products for Unique Needs
The BD special order program allows customers to configure
BD flow cytometers and cell sorters to fit precise research
and assay needs. Tailored to the special needs of researchat the leading edge of biomedical discovery, the program
offers a wide range of choices to help researchers create
the ultimate customized instrument for their requirements.

Multiple Power Options
Innovative laser options include the choice of solidstate lasers across the full spectrum. Currently, 18 laser
wavelength options are offered, ranging from ultraviolet
to infrared wavelengths. Through the special orderprogram, researchers can configure a BD LSRFortessa withup to 7 lasers and 42 positional choices for the detectors.
Forward Scatter PMT Option
The special order program offers a Forward Scatter(FSC) PMT Option that complements small particle
scatter detection. This option lowers the threshold on
size measurements, which is particularly valuable formicrobiology applications.
Evolving Capabilities
BD LSR customers have an expanded level of flexibility
and choice because they can add lasers after purchasinga system, to meet new or evolving requirements. This
flexibility makes BD LSR analyzers a solid, long term
investment.

Optimization of Fluorochrome Excitation
These experiments demonstrate the versatility of the 10-laser special
order BD LSR II cell analyzer. Providing the user choices for laser
excitation wavelength, this system adapts to unique user defined
fluorochromes, for optimization of multicolor experiments.

3

10

4

10

10

5

-334

5

10
3

10

2

-10 0 102
2

2

-10 010

10

3

10

4

10

5

GR1-A594-0610

-255

2

GR1-A594-0610

0 10

2

2

-10 010

-223

0

-664

-375

10

4

CD11b-PE-YG585

10
10

3

10

4

CD11b-PE-G585

10
10

3

10

4

CD11b-PE-B585

CC

5

BB

5

AA

-375

2

-10 010

2

3

10

10

4

5

10

GR1-A594-0610

Effect of laser excitation on spectral overlap
Cells co-stained with Alexa Fluor® 594 (GR1) and PE (CD11b) wererun using blue 488-nm (B), green 532-nm (G), and yellow-green
561-nm (YG) laser as alternate excitation sources for PE. Alexa
Fluor® 594 was excited using an orange 592-nm laser. Panel A
shows PE excitation with a 488-nm laser, panel B shows excitation

12

with a 532-nm laser, while panel C shows excitation with a
561-nm laser. The 10-laser special order BD LSR II instrument allows
the user to choose the excitation source as well as the excitation
power. Researchers can maximize signal while minimizing thecompensation required to deal with unwanted spectral overlap.

0

Count

An Innovation Process
A vigorous pursuit of the latest and best laser technologies
ensures an unparalleled range of configuration choices
offered by the BD special order program for BD LSR
analyzers. New technologies are regularly incorporated into
the product line as soon as they become available.

500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500

CONFIGURATION

102

103

105

FSC-H

1 µm

500 nm

200 nmFigure 1: BD LSRFortessa Standard FSC

1,500
1,000

P1

P2

P3

0

500

Count

2,000

2,500

The ever-expanding list of available lasers demonstrates
BD’s ongoing commitment to perpetual innovation. This is
one of the many ways BD ensures that the BD LSR analyzer
platform continues to support the evolving needs ofleading researchers around the world.

0

101

102

103

104

FSCPMT-H

105

1 µm

500 nm

200 nmFigure 2: BD LSRFortessa Special Order FSC PMT Option
The BD LSRFortessa FSC (data shown in Figure 1) is optimized fora wide range of biological particles. For applications requiring
smaller resolution, a FSC PMT Option is available through the special
order program.

B

150

CFP-CFP 403nm 40mW

C

CFP-CFP 403nm 100mW

100
75

Count

60
50
30

50

40

Count

100

102

103

VBG 470-H

104

105

102

103

VGG 470-H

104

105

0

0

10

25

20

50
0

Count

70

80

150

90 100

CFP-CFP 448nm 40mW

125

A

102

103

VBG 470-H

104

105

Excitation of ECFP using two different excitation wavelengths
Panel A shows data generated using a 448-nm excitation source at
40 mW, Panel B shows a 403-nm excitation source at 40 mW, andPanel C shows a 403-nm excitation source at 100 mW. While ECFP is

optimally excited at 448 nm, it is apparent that an increase in laser
power can largely compensate for a suboptimal excitation source.
Green peak = ECFP-negative cells, aqua peak = ECFP-positive cells.
Data courtesy of A. Smith, R. Salomon, S. Allen, and B. Roediger of the Centenary
Institute, University of Sydney, Sydney, Australia.

13

SERVICES

Services
BD Biosciences is fully committed to the success andsatisfaction of its customers. Supporting flow cytometry
applications for over 35 years, BD has training, technicalapplications support, and field service teams dedicated tohelping customers achieve optimal instrument performance,ease of use, and streamlined workflow. With unmatchedflow cytometry experience, this world-class service
organization is available to help with your BD LSR productinstallation, future upgrades, and application support.

Training
Hands-on training is included with each BD LSRFortessa
analyzer. Courses are held at BD training centersworldwide. BD flow cytometry training courses combine
theory and practice to provide participants with the skills
and experience they need to take full advantage of thecapabilities of their BD LSRFortessa analyzer.
Technical Application Support
BD Biosciences technical applications support specialists areavailable to provide field- or phone-based assistance andadvice. Expert in all aspects of flow cytometry, BD technicalapplication specialists are well equipped to address customer
needs in both instrument and applications support.
Field Service
When instrument installation or service is required, a
BD Biosciences Technical Field Service Engineer can bedispatched to the customer site. BD Biosciences field service
engineers are located across the world. On-site service andmaintenance agreements are available to provide long-term
support for BD LSR analyzers.
Special Order Program
Instruments can be customized to meet customer
requirements via the special order program.

14

Regional Offices
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