Attune NxT Flow Cytometer

Flow cytometry

Efficient. Flexible. Transformative. instrument

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Contents
Explore the Attune NxT Flow Cytometer

Designed for efficiency

Speed and accuracy 4
Smooth flow delivery for accurate counts 6
Reduce clogging from difficult samples 8
Application highlight
Minimize time with no-wash, no-lyse protocol for
blood samples 10
Precision optical performance 12
Attune NxT Autosampler
For even more efficiency 14
Flexibility to create a practical instrument
Detect the full range of fluorescence 16
Expand the range of performance for your violet laser 18
Attune NxT Software
Feature-rich, researcher-inspired software that performs
to your specifications 20
Synthetic biology solutions—CRISPR 22
Stem cell solutions 23
Academic research solutions
Fluorescent proteins 24
Microbiology research solutions
Bacterial analysis 26
Oncology research solutions
13-color human lymphocyte immunophenotyping pane 27
Immuno-oncology research solutions 28
Flow cytometry reagents 29
Robotic automation solutions
Orbitor RS Microplate Move 30
Transforming capabilites
Options that resonate 32
Aftermarket care
Partner with a flow cytometry company invested in
supporting you through a lifetime of research 34
Designed for efficiency
Speed and accuracy

Technology 1. Acoustic-assisted hydrodynamic focusing

With acoustic-assisted hydrodynamic 2. Flat-top lasers

focusing, the Attune NxT Flow

Cytometer (Figure 1) avoids
3. Attune NxT Software
compromise between data quality and
higher sample rates by uncoupling
cell alignment from sheath flow.
Acoustic-assisted hydrodynamic
focusing precisely aligns cells using
ultrasonic radiation pressure (>2 MHz)
to transport particles into the center of
the sample stream. This prefocused
stream is then injected into the sheath
5. Volumetric fluidics
stream (Figure 2). This results in a
4. Fluid storage 6. Autosampler
narrow particle stream and uniform
laser illumination, regardless of the Figure 1. The Attune NxT Flow Cytometer components. (1) Patented acoustic-assisted
sample input rate. hydrodynamic fluidics increase sample input speed while maintaining data integrity. (2) Flat-top
lasers deliver more even application of light to each cell. (3) Invitrogen™ Attune™ NxT Software
designed to guide users through complex flow cytometry experiments. (4) Fluid storage designed
The instrument’s speed for minimal waste. (5) Volumetric fluidics provides cell counting and a resistance to clogging.
specifications include: (6) Autosampler provides easy 1-click transition from tube to plate.
• Sample input flow rate ranges from
12.5 to 1,000 µL/min
• Data acquisition speed up to A B
35,000 events/second with Low sample rates (10–20 µL/min) High sample rates (100–1,000 µL/min)
Hydrodynamic core Hydrodynamic core
34 parameters, based on a
10% coincidence rate per
Laser Laser
Poisson statistics (cross-section) (cross-section)

• Maximum electronic speed

ath
ath

She
She

is 65,000 events/second with

She
She

ath
ath

all parameters Acoustic Acoustic

Sheath

Sheath
Sheath

Sheath

field field

Traditional Acoustic-assisted Traditional Acoustic-assisted

hydrodynamic focusing hydrodynamic focusing hydrodynamic focusing hydrodynamic focusing

Figure 2. Acoustic focusing vs. traditional hydrodynamic focusing as particles pass through
the laser. (A) In acoustic focusing, cells remain in tight alignment even at higher sample rates,
resulting in less signal variation and improved data quality. (B) In traditional hydrodynamic focusing,
increasing the sample rate results in widening of the sample core stream, resulting in increased
signal variation and compromised data quality.

4
Benefits
• Greater reproducibility and consistency in data 35,000
• Maintain consistent concentration results across all flow events/sec
rates (Figure 3)
• Process very dilute or concentrated samples while Up to
maintaining low coefficient of variations (CVs) (Figure 4)
1,000 μL/min
Data
241
12.5 μL/min
300
25 μL/min
400
100 μL/min
10x
No. of events

200 CV = 2.99% CV = 3.03%

300
CV = 2.76% faster*
200
200
100
100 100
A Measured concentration of B Measured concentration of
250 500 250 500 250 500 10 µm beads as a function of rate 3 µm beads as a function of rate
10 6 10 6
PI (x 103) PI (x 103) PI (x 103)
Concentration (beads/mL)

Concentration (beads/mL)
264 292 300
200 μL/min 500 μL/min 1,000 μL/min
No. of events

CV = 2.94% CV = 2.70% CV = 2.96%

10 5 10 5
200 200 200

100 100 100 10 4 10 4

Expected concentration (beads/mL): Expected concentration (beads/mL):

10,000 100,000 1,000,000 10,000 100,000 1,000,000
250 500 250 500 250 500 10 3
10 3

PI (x 103) PI (x 103) PI (x 103) 12.5 25 100 200 500 1,000 12.5 25 100 200 500 1,000
Flow rate (µL/min) Flow rate (µL/min)
Figure 3. Minimal data variation at high sample rates with the
Attune NxT Flow Cytometer. Jurkat cells were fixed and stained with Figure 4. Data demonstrating the measured vs. expected
propidium iodide, treated with RNase, and analyzed at a concentration concentration as a function of flow rate. (A) Measured concentration
of 1 x 10 6 cells/mL at different sample rates. The left peak in all graphs of 10 μm beads as a function of rate. Larger particles (e.g., 10 μm)
reflects cells in G0/G1 phase, while the right peak reflects cells in show consistent results across the flow rate range 100–1,000 μL/min.
G2/M phase. Regardless of sample rate, the width of the G0/G1 and (B) Measured concentration of 3 μm beads as a function of rate. Smaller
G2/M peaks, and the CVs remain consistent, even at the highest sample particles (e.g., 0.2–3 μm) show consistent concentration results across all
rate of 1,000 μL/min. flow rates for the three concentrations of beads/mL tested.

“The ability to run very dilute samples is quite amazing

and might be a life saver on many occasions where
you have little-to-no sample left.”

– J. P. Robinson, PhD
Purdue University

* Than traditional hydrodynamic focusing systems.

5
Smooth flow delivery for accurate counts

The Attune NxT Flow Cytometer delivers samples into the instrument with
minimal variation (Figure 5). Smoother delivery of samples provides more
confidence when
Peristaltic presenting
Pump vs. Positivecell counting Syringe
Displacement data.

A Time vs. count plots showing a moving Data

average of the number of events
A 1,000 B 1,000
1,500
1,000 1,000

(x3)10 3)
(x3)10 3)

(x 10
(x 10

scatter
scatter
1,000 500 500
Lymphocytes

scatter
Count

scatter
Cell counting 500 91.9 500
Lymphocytes
CD3 –: 29.6 CD3 +: 62.2

SideSide
SideSide
spread 91.9
500 CD3 –: 29.6 CD3 +: 62.2
Peristaltic Pump vs. Positive Displacement Syringe 0 0
10 2 10 3 10 4 10 5 0 10 4
10 5 10 6
0 0
CD45
10 2 Pacific Orange
10 3 fluorescence
10 4 (VL1)
10 5 CD3
0 APC 10
fluorescence
4 (RL1)
10 5 10 6
0 CD45 Pacific Orange fluorescence (VL1) CD3 APC fluorescence (RL1)
0 2 4 6 8 10 12 14 16
10 6 10 6
CD8: 16.9
Time (10 6) C 10 65
CD8: 16.9
D 10 65
CD19: 8.9

(VL1)
Blue
CD19: 8.9
(YL4)

10 5 10 5

(VL1)
Blue
B
PE-Cy7
(YL4)

Time vs. count plots showing a moving

fluorescence
Pacific
10 4
fluorescence

average of the number of events CD4: 42.0

PE-Cy7

fluorescence
Pacific
10 4 CD56: 17.2
10 4
fluorescence

CD4: 42.0
1,500
CD8CD8

10 4 CD56: 17.2

CD19
0 0

CD19
0 0
0 10 4 10 5 10 6 –10 3 0 10 3 10 4 10 5 10 6
1,000
CD4 Alexa
0 Fluor 488
10 4fluorescence
10 5 (BL1)
10 6 –10 3 CD56
0 PE10
fluorescence
3
10 4 (YL1)
10 5 10 6
Count

CD4 Alexa Fluor 488 fluorescence (BL1) CD56 PE fluorescence (YL1)

Cell counting
500 spread

0
0 2 4 6 8 10 12 14 16 +

Time (10 6)
-

Figure 5. Time vs. count plot to obtain a moving average of the

number of events showing up at a given time. (A) Data from a flow
cytometer with a peristaltic pump, showing fluid pulsation ±33% of the
average count and a total spread of 66% of the average count. (B) Time Figure 6. Lymphocyte subset analysis. A 100 μL aliquot of normal
vs. count data from Attune NxT Flow Cytometer with a non-peristaltic human whole blood was labeled with fluorophore-conjugated antibodies
pump, showing fluid pulsation of ±5%. against CD surface markers, followed by red blood cell (RBC) lysis
using 2 mL of Invitrogen™ High-Yield Lyse Fixative-Free Lysing
Solution (Cat. No. HYL250), resulting in a 1:21 dilution of the blood.
Technology (A) Lymphocytes are identified on a density plot of CD45 vs. side scatter
Samples on the Attune NxT Flow Cytometer are delivered with an oval gate around the lymphocyte (CD45+) population. (B) Cells
by a positive-displacement syringe pump for volumetric in the lymphocyte gate are displayed on a density plot of CD3 vs. side
scatter. Rectangle gates surround the CD3+ T cell and CD3 – B and
analysis meaning that all events are automatically natural killer (NK) cell populations. (C) Cells in the CD3+ gate are then
counted, and particle counts or concentrations can displayed on a density plot of CD4 vs. CD8 to quantify CD4+ helper T
be viewed with the simple click of a button. Figure 6 cells (CD4+ CD3+ CD45+) and CD8+ cytotoxic T cells (CD8+ CD3+ CD45+).
(D) CD3 – cells are displayed on a density plot of CD56 vs. CD19 to
shows the scatter plots and cell concentrations for all distinguish CD56+ NK cells from CD19+ B cells. The statistics table shows
lymphocyte subpopulations. the gating and measured concentrations (cells/μL).

6
Benefits
• Syringe easily removed for cleaning or replacement Tips
• Consistent cell concentration results across all flow rates Useful for samples that are inherently low
(Figure 7) in concentration, such as cerebrospinal
fluid (CSF), and stem cell samples with low
• Precise counts without the need for expensive beads
cell numbers.

25
100 µL/min
200 µL/min
20
500 µL/min
Concentration (cells/µL)

15

10

0
CD4+ CD8 + CD19+ CD56 +

Figure 7. Replicate samples collected at three flow rates on the

Attune NxT Flow Cytometer. Cell concentrations were measured
using three different flow rates: 100, 200, and 500 μL/min. The Attune
NxT Flow Cytometer provides similar concentration measurements for
each lymphocyte subpopulation, regardless of the flow rate. Each bar
represents the mean cells/μL ±standard deviation of three samples run at
each indicated flow rate for each population.

7
Reduce clogging from difficult samples

Your research samples are precious as they are often

difficult to produce. The Attune NxT Flow Cytometer is less
Tips
prone to clogging, allowing challenging samples such as The higher the flow rate a sample is run on
cardiomyocytes, heterogeneous blood cells, and cancer the Attune NxT Flow Cytometer, the lower
cells to flow with confidence. the amount of sheath fluid that is used.

Technology
Engineered to actively resist clogging, a syringe-driven
system (Figure 8) and larger flow cell help prevent the
loss of precious sample such as cancer stem cells from
primary pancreatic tumors (Figure 9), and is drastically “We have yet to clog the machine with our debris-rich
less susceptible to clogs. The Attune NxT Flow Cytometer primary tumor samples. Of course, the acoustic
employs a non-pressurized system that mechanically technology greatly facilitates the identification of small
decreases the occurrence of clogging. populations, like cancer stem cells, increasing our
capacity to detect and quantify these rare events with
Benefits high efficiency and reliability.” 
• Easy flow of difficult samples such as large or sticky cells
– Bruno Sainz Jr, PhD
• Sample recovery feature built into software
Autónoma University of Madrid, School of Medicine
• Comparatively lower fluid consumption (~1.8 L/day)

Focusing fluid
reservoir

Valve

1 mL sample
syringe

Figure 8. Positive-displacement syringe pump. Syringe easily

removed for cleaning or replacement.

8
 Data

A B
1M 1M
10
1M5

10 5
800K 800K
800K
104
104
600K 600K

SSC-A
FSC-H

DAPI
600K

FSC-H

DAPI
10 3
10 3
400K 400K
400K

200K 10 2 200K2
200K 10
0
0
0 0
0 200K 400K 600K 800K 1M 0 0 200K 400K 600K 800K 1M 0 200K 400K 600K 800K
0 200K 400K 600K 800K 1M 0 200K 400K 600K 800K
Ungated FSC-A Single FSC-A Live FSC-A
Ungated FSC-A Single FSC-A

C 1M D 1M
1M 1M 10
1M5
1M
10 5
800K 800K

Autofluorescence
800K 800K 800K
800K 104
10
600K
4
600K
SSC-A

SSC-A

600K 600K 600K

SSC-A

SSC-A
SSC-A

600K
SSC-A

10 3
DAPI

400K
10 3 400K
400K 400K 400K
400K
200K 200K 10 2
200K
10 2 200K 200K
200K 0
00 0
0 0 10 2 10 3 104 10 5 0 0 10 2 10 3 104 10 5 0 0 10 2 10 3 104
0
1M 0 200K 400K 600K 800K 1M 0 10 2 10 3 104 10 5 0 10 2 10 3 104
1M 0 200K 400K
EpCAM-APC 600K 800K 1M 0 200K 400K
CD90-APC600K 800K 1M Autofluorescence
Debris-free Debris-free Debris-free
Live FSC-A Debris-free EpCAM-APC Debris-free CD90-APC
Single FSC-A Live FSC-A

E F
1M
10 5
10 5

800K
Autofluorescence

Autofluorescence

104
104
600K
SSC-A

10 3
10 3
400K

10 2
200K 10 2
0
0
0
10 55 0 10 22 10 33 1044 10 55
10 0 10 10 10 10 0 10 2 10 3 104 10 5
Debris-free Autofluorescence
CD90-APC Autofluorescence
Debris-free Debris-free

Figure 9. The Attune NxT Flow Cytometer detects autofluorescent and CD90+ rare cancer stem cells from primary pancreatic tumors
without clogging. Tumors were minced and enzymatically digested with collagenase, followed by an overnight incubation with 30 μM riboflavin in
RPMI medium with 10% FBS. Cells were then blocked with flebogamma and stained with anti-EpCAM or anti-CD90 antibodies. (A–C) Single, live,
and debris-free cell gating strategy. (D) EpCAM+, (E) CD90+, and (F) autofluorescence-positive cells within the tumor population. Data courtesy
Bruno Sainz Jr, PhD.

9
Application highlight
Improved data resulting from less trauma to cells

Acoustic focusing allows the Attune NxT Flow Cytometer

“Multiplexing and compensation are much easier and
to deliver a no-wash, no-lyse protocol (Figure 10) to
extremely efficient with the Attune NxT (cytometer).” 
minimize cell loss, significantly reduce time, and simplify
sample preparation. – Bruno Sainz Jr, PhD
Autónoma University of Madrid, School of Medicine
Benefits
• Improve lab safety with reduced sample handling with
no-wash protocol
• Completely cut out time-consuming centrifugation steps
• Save countless hours running dilute samples and reduce
reagent costs
• Eliminate cell loss due to wash steps or RBC
removal procedure
• Ideal for limited sample volumes and for functional
live-cell assays (Figure 11)

Protocol

Generic sample preparation workflow

Single-cell Wash Lyse Wash Stain Wash

suspension (5–10 min) (30–60 min) (5–10 min) (15–30 min) (5–10 min) Analyze
(5–10 min)

65–130 min

No-wash, no-lyse sample preparation workflow

Up to
Single-cell

65%
Stain Analyze
suspension (15–30 min) Dilute 10x faster*
(5–10 min)
reduction in
prep time
20–40 min

* Compared to conventional cytometers.

Figure 10. No-wash, no-lyse sample preparation workflow.

10
Data

All events All events PLT + WBC

A B C
WBCs: 5.7%
VL1-H - violet 405 nm SSC

VL1-H - violet 405 nm SSC

PLT + WBC: 4.7%

SSC-H blue 488 nm

Platelets: 93.8%

RBC: 93.8%

Noise: 1.3%
SSC-H blue 488 nm SSC-H blue 488 nm FSC-H

All events All events

D E
VL1-H - Violet 405 nm SSC

WBCs: 5.7%
VL1-H - violet 405 nm SSC

PLT: 4.4%

RBC: 93.8%

Noise1: 1.3%

SSC-H blue 488 nm SSC-H blue 488 nm

Figure 11. Forward scatter (FSC) and side scatter (SSC) analysis with blue (488 nm) and violet (405 nm) lasers on intact whole blood (no-
lyse, no-wash). (A, B) RBCs, white blood cells (WBCs), and platelets are separated on the basis of light scatter only by using a combination of blue
and violet laser SSC analysis. Hemoglobin in RBCs readily absorbs light at 405 nm, shifting the RBC population to the right by reducing the SSC for
RBCs in the violet laser channel relative to leukocytes and platelets. Dual FSC and SSC threshold is set low enough to show instrument noise, ensuring
the full platelet population is visualized. (C) Using the gate that includes WBCs and platelets, a standard plot of FSC vs. 488 nm SSC can be used
to distinguish the platelet population from the WBCs with regions created around the two populations. (D) Using color-backgating on plot (A), the
RBC population is colored red, the platelet population is colored green, and the WBC population is colored blue, while the noise is black. The three
main WBC populations of lymphocytes, monocytes, and granulocytes can be distinguished. (E) Placing regions around the RBC, WBC, and platelet
populations show the dominant cell type in whole blood is the RBC, while the WBCs and platelets are relatively rare events.

11
Precision optical performance

Minimize instrument downtime with the Attune NxT optical Technology

system. The Attune NxT lasers are designed to last the The Attune NxT Flow Cytometer uses flat-top lasers with
life span of a flow cytometer and provide a wider area of an intensity profile that allows a much wider window of
light intensity. alignment (Figure 12). This innovative design helps ensure
precise fixed alignment of 4 spatially separated solid-state
lasers onto the sample stream (Figure 13), minimizing the
effects of changes in fluidics or optics. The stability of the
optical system leads to increased data consistency over
time, superior performance, and first-class reliability.

A B
CV = 1.1 CV > 8

Gaussian: aligned Gaussian: misaligned

C D
CV = 1.1 CV = 1.7

Flat-top: aligned Flat-top: still aligned

Figure 12. Emission profiles of lasers used in flow cytometers. Figure 13. The Attune NxT Flow Cytometer can be configured with
(A) Gaussian laser profile with proper alignment, (B) Gaussian laser up to 4 spatially separated lasers.
profile with misalignment, (C) flat-top laser profile with proper alignment,
and (D) flat-top laser profile still in proper alignment.

12
Benefits
• No warm-up delay: fiber isn’t affected by instrument “Having evaluated the instrument over several months,
warm-up I would say the Attune NxT Flow Cytometer fits the
• Simmer mode: automatic shutoff prolongs laser usage superior category of flow cytometers.”
lifetime up to 10x – J. P. Robinson, PhD
• Lasers are only turned on when acquiring samples Purdue University

13
Attune NxT Autosampler
For even more efficiency

Improve workflow efficiency with the high-throughput

option—the Invitrogen™ Attune™ NxT Autosampler. Built-in “The dual tube-to-plate operation—instant change from
compatibility switches between tubes and plates with tubes to plates is really an excellent feature.”
a single click in Attune NxT Software. The Attune NxT
– J. P Robinson, PhD
Autosampler is compatible with many different plate
Purdue University
formats, including 96-well, 384-well, and deep-well plates.
The system is designed to provide minimal variation
regardless of sampling method (tube vs. plate) and
collection rate (Figures 14 and 15).

Technology

Acquisition time*
• <42 min for 96-well plate
• <180 min for 384-well plate
Carryover
• <0.5% in “Plate Loader” format—standard mode,
2 wash cycles
• Ultralow carryover—multiple rinse capability
Extended fluidics option
• Optional external fluid tank with 10 L fluid capacity.

* Using one rinse and one mix (aspiration) and full analysis of a 40 μL sample.

Benefits
“We looked at several metrics and compared the
• One-click transition from tubes to plates using
Attune NxT Autosampler to other 96-well plate readers.
Attune NxT Software
The autosampler proved to have very good stability
• Performs automated cleaning when the instrument is and very low carryover. We were most impressed
shutting down by the way that the autosampler took advantage of
• Mixes sample by aspiration instead of shaking, ensuring the Attune NxT Flow Cytometer’s fluidics and high-
homogeneity of the sample and maintenance of volume throughput. Without compromising stability or
cell viability precision, the autosampler was able to run plates much
faster than any other plate reader.”

– E. M. Meyer
University of Pittsburgh Cancer Institute

14
 Autosampler
Autosampler
Tubes
Tubes
and
and
Plates
Plates

Data
A Tube B Plate
106 106 106 106

CD8 R -PE fluorescence

CD8 R -PE fluorescence

CD8 R -PE fluorescence

CD8 R -PE fluorescence

10 5
10 5
10 5
105

104 104 104 104

103 103 103 103

-103 -103 -103 -103

-103 103 -103 103 104 10

10
4 5
106105 106 -103 103 -103 103 104 1045 106105 106

CD4 FITC
CD4fluorescence
FITC fluorescence CD4 FITC
CD4fluorescence
FITC fluorescence

Figure 14. Consistent results are achievable regardless of sampling method. Whole blood lysed with ammonium chloride was labeled with
Invitrogen™ mouse anti–human CD45 Pacific Orange™, mouse anti–human CD4 FITC, and mouse anti–human CD8 R-PE antibody conjugates. Labeled
samples were analyzed on a blue and violet laser–configured Attune NxT Flow Cytometer equipped with a 488 nm laser for fluorescence excitation of
FITC (530 BP) and R-PE (574/24 BP), and a 405 nm laser for Pacific Orange dye (603/48 LP). Identical samples, including compensation controls, were
analyzed using either (A) tube mode or (B) plate mode with a standard collection rate of 200 μL/min. Lymphocytes were gated using a CD45 vs. side
scatter plot and analyzed for expression of CD4 and CD8 antigens. Minimal variation was observed between analysis in a tube alone and on a plate
running on the Attune NxT Autosampler.

A B

Figure 15. Consistent well-to-well results: the Attune NxT Autosampler heat-map function identifies variation within a parameter across a
96-well plate. Live and heat-killed THP-1 cells were stained with 2 μg/mL propidium iodide, dispensed into a 96-well V-bottom plate, and run at a
standard collection rate of 500 μL/min with 2 mix cycles per well and 2 rinse cycles between wells. Propidium iodide was excited using a 488 nm laser
(640 LP). (A) On the heat map, a color gradient graphically represents the percentage of propidium iodide–positive cells (dead cells). Red-colored
wells indicate 0% propidium iodide–positive cells (live cells) within the sample analyzed from that well; magenta-colored wells indicate a sample
containing 100% propidium iodide–positive cells. (B) The values overlaid on each well in the heat map are the measured percentages of dead cells in
the individual wells. Minimal variation is observed in propidium iodide fluorescence across each row of the entire plate, with a CV of 1.44% for the entire
data set (96 wells).

15
Flexibility to create a practical instrument
Detect the full range of fluorescence

The Attune NxT Flow Cytometer accommodates up to

14 color panels. The filter and laser are configurable and
field upgradable, giving the freedom to upgrade up to 4
lasers and 16 detection channels (Tables 1 and 2).

Table 1. The Attune NxT Flow Cytometer system configurations.

Total
Laser Violet Blue Yellow Green Red detection
Lasers configuration Cat. No. 405 nm 488 nm 561 nm 532 nm 637 nm channels*
Available as Available as Available as Available as
Blue A24864 4 6
1 upgrade upgrade upgrade upgrade
Available as Available as
Blue/green A28995 3 – 4 9
upgrade upgrade
Available as Available as
Blue/yellow A24861 3 4 – 9
upgrade upgrade
Available as Available as Available as
2 Blue/red A24863 4 3 9
upgrade upgrade upgrade
Available as Available as Available as
Blue/violet A24862 4 4 10
upgrade upgrade upgrade
Available as Available as
Blue/violet 6 A29002 6 3 – 11
upgrade upgrade
Available as
Blue/green/red A28997 3 – 4 3 12
upgrade
Available as
Blue/red/yellow A28993 3 4 – 3 12
upgrade
Blue/green/ Available as
A28999 4 3 – 4 13
3 violet upgrade
Blue/violet/ Available as 13
A24859 4 3 4 –
yellow upgrade
Available as Available as
Blue/red/violet A24860 4 4 3 13
upgrade upgrade
Available as
Blue/red/violet 6 A29003 6 3 – 3 14
upgrade
Blue/red/violet
A29001 4 3 – 4 3 16
/green
Blue/red/yellow
4 A24858 4 3 4 – 3 16
/violet
Blue/red/yellow
A29004 6 2 3 – 3 16
/violet 6

* Includes forward scatter (FSC) and side scatter (SSC).

16
 Up to 16 parameters
Benefits
• Field upgradeability to accommodate expanding needs
• Use more lasers for expanded multicolor panel design options
Up to 4 lasers

• Use less reagents

Up to 14 colors

Table 2. The Attune NxT Flow Cytometer filter configurations.

Cat. No. A24864 A28995 A24861 A24863 A24862 A29002 A28997 A24860 A28999 A28993 A24859 A29003 A29004 A29001 A24858

Detectors 4 7 7 7 8 9 10 10 11 10 11 12 14 14 14

Channel Emission filter (nm)

BL1 530/30 525/50 530/30 530/30 530/30 530/30 525/50 530/30 525/50 530/30 530/30 530/30 530/30 525/50 530/30

BL2 574/26 590/40 590/40 574/26 574/26 574/26 590/40 574/26 590/40 574/26 590/40 574/26 695/40 590/40 590/40

BL3 695/40 695/40 695/40 695/40 695/40 695/40 695/40 695/40 695/40 695/40 695/40 695/40 695/40 695/40

BL4 780/60 780/60 780/60 780/60

GL1 575/36 575/36 575/36 575/36

GL2 620/15 620/15 620/15 620/15

GL3 695/40 695/40 695/40 695/40

GL4 780/60 780/60 780/60 780/60

YL1 585/16 585/16 585/16 585/16 585/16

YL2 620/15 620/15 620/15 620/15 620/15

YL3 695/40 695/40 695/40 780/60 695/40

YL4 780/60 780/60 780/60 780/60

RL1 670/14 670/14 670/14 670/14 670/14 670/14 670/14 670/14

RL2 720/30 720/30 720/30 720/30 720/30 720/30 720/30 720/30

RL3 780/60 780/60 780/60 780/60 780/60 780/60 780/60 780/60

VL1 440/50 450/40 440/50 440/50 440/50 450/40 450/40 440/50 440/50

VL2 512/25 525/50 512/25 512/25 512/25 525/50 525/50 512/25 512/25

VL3 603/48 610/20 603/48 603/48 603/48 610/20 610/20 603/48 603/48

VL4 710/50 660/20 710/50 710/50 710/50 660/20 660/20 710/50 710/50

VL5 710/50 710/50 710/50

VL6 780/60 780/60 780/60

Figure 16. The optical filters in the Attune NxT Flow Cytometer
are user-exchangeable, easily slotted in and out of the optical
bench to maximize your capacity.

17
Expand the range of performance for your violet laser

The Attune NxT Flow Cytometer is easily upgradable Benefits

to 6-channel detection for the violet (405 nm) laser • Modular expansion options for growth when needed,
(Table 3). The Attune NxT Flow Cytometer with violet not before
6 channel configuration is designed to accommodate • Facilitate application development with fewer restrictions
a wide variety of experimental conditions. Combined on fluorochrome detectors
with the Invitrogen™ Super Bright and other appropriate
• Enhanced capability to perform a variety of applications
dyes, the system provides expanded choices for panel
on a single instrument
design (Table 4). See available Super Bright dyes at
thermofisher.com/superbright Download the poster at: thermofisher.com/attune-14C

Technology

Table 3. Attune NxT Flow Cytometer configuration All events

10 6
using 6 fluorescence detectors for the violet laser.
Fluorescence detectors
Laser
2-laser 3-laser 4-laser 10 5
SSC-H
Violet, 405 nm 6 6 6
Blue, 488 nm 3 3 2
104
Yellow, 561 nm NA NA 3
Red, 637 nm NA 3 3
Total fluorescence detectors
9 12 14 10 0
available 10 0 101 10 2 10 3 104 10 5 10 6
Total parameters per FSC-H
11 14 16
configuration*
* Includes FSC and side scatter SSC. Figure 17. FSC and SSC discrimination of 0.2 µm, 0.5 µm, and
0.8 µm particles using the Submicron Bead Calibration Kit from
Bangs Laboratory.

Table 4. Fluorophore guidelines for the 6 fluorescence detectors off the violet laser in the Attune NxT
Flow Cytometer.
Detector Bandpass (nm) Fluorophores*
Super Bright 436, eFluor 450, LIVE/DEAD™ Fixable Violet, Vybrant™ DyeCycle™ Violet,
VL1 450/40 SYTOX™ Blue, CellTrace™ Violet, VioBlue™, Brilliant Violet™ 421, Pacific Blue™, BD
Horizon™ V450
eFluor 506, LIVE/DEAD™ Fixable Aqua, CFP, VioGreen™, Brilliant Violet™ 510, Pacific
VL2 525/50
Green™, BD Horizon™ V500
Super Bright 600, LIVE/DEAD™ Fixable Yellow, Qdot™ 605, Pacific Orange™, Brilliant
VL3 610/20
Violet™ 605
VL4 660/20 Super Bright 645, Brilliant Violet™ 650
VL5 710/50 Super Bright 702, Qdot™ 700, Brilliant Violet™ 711
VL6 780/60 Super Bright 780, Brilliant Violet™ 786
* List is not inclusive of all available fluorophores.

18
Violet laser for small-particle detection
The Attune NxT Flow Cytometer can be configured
with optional violet side scatter for better small-particle
resolution. With optimal fluorescence sensitivity and up
to 16-parameter detection capability on particle sizes as
small as 0.2 µm (Figure 17), the Attune NxT Flow Cytometer
supports a large variety of multiparameter applications
(Figure 18).

Data

A All events B Live cells C Singlet 1 D Singlet 2

SSC-A (10^3)

SSC-A (10^3)
SSC-A (10^3)

FSC-A (10^3)

Viability-Fixable Near-IR—RL3 FSC-H (10^3) SSC-H (10^3) CD3-Super Bright 645—VL4

E CD4+ F CD4+ G Live CD3 + L All events

CD45RA PerCP-Cy5.5—BL2

CD4-Super Bright 600—VL3

CD25 Super Bright 702—VL5

Live cells
Singlet 1
Singlet 2
Live CD3+
CD4+
CD25+CD127— (Treg)
CD45RA—CD196—
CD194—CD183+ (Th1)
CD194+CD183— (Th2)
CD127 Brilliant Violet 510—VL2 CD196 Super Bright 436—VL1 CD8 Alexa FLuor 700—RL2 CD45RA—CD196+
H CD45RA CD196
- — I CD45RA CD196
- + J K CD194+CD183— (Th17)
CD45RA+CD196—
CD4 +CD278 + (ICOS) CD8 +CD278 + (ICOS)
CD183 eFluor 600—RL1

CD183 eFluor 600—RL1

CD4+CD223+
CD4 +CD134 + (OX40) CD8 +CD134 + (OX40)
CD4 +CD279+ (PD-1) CD8 +CD279+ (PD-1) CD4+CD278+
CD4 +CD223 + (LAG-3) CD8 +CD223 + (LAG-3) CD4+CD134+
CD4+CD279+
CD8+
CD8+CD223+
CD8+CD278+
CD8+CD134+
CD194 PE-Cy7—YL3 CD194 PE-Cy7—YL3 CD8+CD279+

Figure 18. T lymphocyte immunophenotyping: 14-color flow cytometry panel design using the Attune NxT violet 6 channel option and
Super Bright fluorescent dyes gating strategy. (A) A region is placed around live peripheral blood mononulcear cells (PBMCs) as identified by the
Invitrogen™ LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit. (B, C) Live cells are analyzed through sequential singlet gating. A region is then placed on
the (D) CD3+ population for gating on (E) CD4+ and CD8+ populations. The CD4+ population is used to gate on (F) CD127 vs. CD25, for (G) CD45RA
vs. CD196, and (J) CD278, CD134, CD279, and CD223 populations. The CD45RA –/CD196 – population from (G) is gated on (H) CD183 vs. CD194. The
CD45RA –/CD196+ population from (G) is gated on (I) CD183 vs. CD194. The CD8+ population from (E) is used for gating (K) CD278, CD134, CD279,
and CD223 populations. (L) The entire gating strategy is displayed in hierarchical format using the Attune NxT violet 6 channel option and v2.6 software
for easy visualization.

19
Attune NxT Software
Feature-rich, researcher-inspired software that performs
to your specifications
The Attune NxT Software was masterfully developed to
offer user-focused functionality with many automated,
user-definable, and administrative features to provide
powerful data acquisition and analysis simple enough for
users at any experience level (Figure 19).

Speed
• Increase productivity with live-streaming update of
statistics during acquisition of events
• Fast refresh rates for large data sets of up to 20 million
events per sample with option to append

Guided functionality
• Automated maintenance: “Startup”, “Shutdown”, “One of the more impressive aspects of the Attune
“Rinse”, “Sanitize Attune™ SIP”, “Deep Clean”, “Sanitize”, NxT Software is its “ease of use”. As a shared facility
“Decontaminate”, “Autosampler Calibration” manager, I instruct a wide variety of users how to run
• “Automated Backup Options” to ensure data redundancy a wide variety of instruments. It is often a challenge
to teach a new, and at times, veteran cytometrist how
• Heat map for easy setup of plate-based assays
to operate a new system. User-friendly software is a
• “Sample Recovery” to return unused sample to save must. Several of my facility users picked up on the
precious samples Attune NxT Software right away. Many were able to
• Hierarchy view of plots to instantly view complex run complex multi-parameter experiments in their first
gating strategies session; some of them were doing this on their own
without any assistance from the facility staff. If only all
• “Autosampler Calibration” of the Attune NxT Autosampler
of our instruments were so easy to use!”
every 30 days to ensure optimal performance
– E. M. Meyer
Customizable
University of Pittsburgh Cancer Institute
• User customizable “Wait-to-Record” function
• Ability to set user options for default settings for gates,
plots, fonts, colors, and group/sample names
• Visual appearance of the plots is completely
customizable; fonts, colors, titles legends, and much
more can appear exactly the way you want

Publication-quality data
• Smart gate naming to customize quad gate names and • Overlay module to perform comparative analysis of
target names single- and dual-parameter data
• Add text, statistics, and even images to make your • Preview plots to instantly view all combinations of
data pop parameters in a file
• 1-click saving of high-resolution plots in a variety of
file formats

20
 Drag and drop
Preview panel to instrument, workspace,
Quick-access toolbar easily determine or compensation
for frequently used which plots to add settings to apply to
commands to workspace Smart gate labeling new experiments

Batch processing
can easily be
accomplished by
moving between
Acquisition status sample-specific
lets you see the workspaces by
event rate and the clicking on the
total number of “WS” indicator for
events collected each sample

Achieve workspace
flexibility by
Easily set run
creating different
parameters
workspaces for
samples, groups, or
an entire experiment

Choose from a Easily monitor usage Further illustrate your

variety of plot types system log analysis with text or
add from a full range
of statistics

Figure 19. Intuitive, user-friendly software interface with familiar workflow.

Compensation tools
• Both negative and unstained gating parameters
are available
• On-plot compensation for fine-tune adjustment
• Modification of compensation to add or remove
parameters as needed after compensation is set up
• Set up and collect compensation controls directly from
a plate

User management
• Levey-Jennings and “Performance History” reports of
baseline and performance tests to monitor trends
Tips
• Ability to create and manage multiple user accounts Need to delay the time between
sample acquisition and when it is
• System access based on user-account privileges recorded from a plate? No problem
with “Wait-to-Record” function.
Learn more about Attune NxT Software at
thermofisher.com/attune-cytometer-software

21
Synthetic biology solutions—CRISPR

Flow cytometry fits into the CRISPR analysis workflow, Data

enabling researchers to monitor the efficiency of genome ssODN
1.0M
editing experiments. Fluorescent protein reporters allow
measurement of transfection rates, optimization of your 800K

conditions, and rapid analysis using flow cytometry. 600K

400K
Technology
200K
The Invitrogen™ GeneArt™ Genomic Cleavage Selection Kit,
0
which enables measurement of the percentage and the 10 0 101 102 10 3 10 4 10 5 10 6

mean fluorescence intensity (MFI) of orange fluorescent GFP

protein (OFP)–expressing cells using a flow cytometer, IVT gRNA + IVT gRNA +
contains a vector with the OFP gene for a quick visual Cas9 mRNA Cas9 mRNA + ssODN
check on the functionality of the engineered nuclease. 1.0M 1.0M

800K 800K

Benefits 600K 600K

When screening libraries or large sample populations
400K 400K
of edited cells, flow cytometry enables razor-precision
200K 200K
analysis. With appropriate antibodies, fluorescent proteins,
0 0
or functional probes, complex phenotypes can be
10 0 101 102 10 3 10 4 10 5 10 6 10 0 101 102 10 3 10 4 10 5 10 6
unraveled through multiplexing (Figure 20).
Figure 20. Tracking blue fluorescent protein (BFP) converting to
• Process optimization using fluorescent protein reporters green fluorescent protein (GFP) by homologous recombination using
allow you to quickly measure transfection efficiencies the CRISPR-Cas9 system. Single-stranded oligodeoxynucleotides
(ssODN) assist in making large genomic changes following cleavage by
• Rapid library screening Cas9 nuclease and in vitro–transcribed guide RNA (IVT gRNA).

• Save time and money using flow cytometry for

genome editing

22
Stem cell solutions
Flow cytometry analysis of transcription factors during
cardiomyocyte differentiation
The ability to direct human pluripotent stem cells (hPSCs) Benefits
toward differentiated cell phenotypes offers tremendous • Accelerate discovery and screening workflows
potential for personalized and regenerative medicine. • Ideally suited for use with fragile and large cell types like
Quantification of the dynamic expression patterns stem cells and cardiomyocytes (Figure 21)
of transcription factors that underlie cardiomyocyte
• Gentle and safe analysis without clogging the instrument
differentiation often relies on detection of mRNA transcripts
or wasting cells
via quantitative reverse transcription PCR (RT-qPCR) in cell
and tissue lysates made from heterogeneous populations Read the BioProbes™ Journal article:
of cells. thermofisher.com/attune-cardiomyocyte

Data
Day 1: Singlet Day 2: Singlet Day 3: Singlet Day 4: Singlet Day 5: Singlet
A B C D E
104 104 104 104 104
Oct4-Alexa Fluor 488-H

Oct4-Alexa Fluor 488-H

Oct4-Alexa Fluor 488-H

Oct4-Alexa Fluor 488-H

Oct4-Alexa Fluor 488-H

103 103 103 103 103

102 102 102 102 102

0 0 0 0 0

-102 -102 -102 -102 -102

10 2
10 3
10 4
10
5
10
6
102
10 3
104
105
106
102
103
10 4
10 5
106
102 103 104 105 106
Nkx2.5-Alexa Fluor 647-H Nkx2.5-Alexa Fluor 647-H Nkx2.5-Alexa Fluor 647-H Nkx2.5-Alexa Fluor 647-H Nkx2.5-Alexa Fluor 647-H

F Day 6: Singlet
G Day 7: Singlet
H Day 8: Singlet
I
Day 9: Singlet
J
Day 10: Singlet

104 104 104 104 104

Oct4-Alexa Fluor 488-H

Oct4-Alexa Fluor 488-H

Oct4-Alexa Fluor 488-H

Oct4-Alexa Fluor 488-H

Oct4-Alexa Fluor 488-H

103 103 103 103 103

102 102 102 102 102

0 0 0 0 0

-102 -102 -102 -102 -102

102 103 104 105 106 102 103 104 105 106 102 103 104 105 106 102 103 104 105 106 102 103 104 105 106
Nkx2.5-Alexa Fluor 647-H Nkx2.5-Alexa Fluor 647-H Nkx2.5-Alexa Fluor 647-H Nkx2.5-Alexa Fluor 647-H Nkx2.5-Alexa Fluor 647-H

Figure 21. Two-parameter plots representing staining profiles for Oct4 and Nkx2.5 in H9 hPSC cells during cardiomyocyte differentiation. All
plots were gated on singlet cells. (A) At day 1, nearly all cells are Oct4+ and Nkx2.5 –, consistent with a pluripotent state. (B–J) During the time course
of differentiation, with data shown for each day of differentiation, cells lose Oct4 expression and begin to express the cardiac marker Nkx2.5. The
precedence-density plot display is used, with the red-colored population representing Nkx2.5+ cells, and the green-colored population representing
Oct4+ cells.

23
Research solutions
Fluorescent proteins

The Attune NxT Flow Cytometer supports a method for Benefits

detecting multiple fluorescent proteins for simultaneous • Achieve effective transfection efficiency detection, and
analysis within the same cell, thus overcoming a broad expression of one to many fluorescent proteins using
emission spectrum and resulting spectral overlap. Excite flow cytometry
and detect GFP and YFP with the same set of laser • No-hassle labeling with ready-to-use kits for fluorescent
and bandpass filters. As shown in Figure 24, with an protein detection (Figure 26)
appropriate filter set, the 488 nm laser efficiently excites
• Simultaneous detection of GFP and YFP
both fluorescent proteins simultaneously. The GFP and
YFP signals can be appropriately discriminated using
the Invitrogen™ Attune™ NxT Fluorescent Protein Filter Kit
(Figure 25).

Data

100 100 100 100

80 80 80 80
Percent of max

60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
101 10 2 10 3 104 10 5 10 6 101 10 2 10 3 104 10 5 10 6 10 2 10 3 104 10 5 10 6 101 10 2 10 3 104 10 5 10 6

TagBFP fluorescence (VL1 440/50) emGFP fluorescence (BL1 530/30) YFP fluorescence (BL1 530/30) mOrange2 fluorescence (YL1 585/16)

100 100 100

80 80 80
Percent of max

60 60 60

40 40 40

20 20 20

0 0 0
10 2 10 3 104 10 5 10 6 101 10 2 10 3 104 10 5 10 6 10 2 10 3 104

TagRFP fluorescence (YL1 585/16) mKate fluorescence (YL2 620/15) mCherry fluorescence (YL2 620/15)

Figure 24. Detection of a palette of fluorescent proteins using the Attune NxT Flow Cytometer. Cells were transfected or transduced with
vectors expressing different fluorescent proteins. Samples were acquired at a flow rate of 100 μL/min using 405 nm, 488 nm, or 561 nm excitation
sources. The gray peaks represent control cells that do not express fluorescent proteins.

24
A 10 6 10 6 B 10 6 10 6
540/30)
540/30)

540/30)
540/30)
10 6 10 6 10 6 10 6
10 5 10 5 10 5 10 5
540/30)
540/30)

540/30)
540/30)
10 5 10 5 10 5 10 5
104 104 104 104
(BL2
(BL2

(BL2
(BL2
104 104 104 104
(BL2
(BL2

(BL2
(BL2
10 3 10 3 10 3 10 3
10 3 10 3 10 3 10 3
fluorescence
fluorescence

fluorescence
fluorescence
fluorescence
fluorescence

fluorescence
fluorescence
10 2 10 2 10 2 10 2
1002 1002 1002 1002
–1002–1002 –1002–1002
–10 2–10 2 –10 2–10 2
YFP
YFP

YFP
YFP
YFP
YFP

YFP
YFP
–10 3–10 3 –10 3–10 3
–10 3–10 3
–10 2–10 2 –10 2–10
0 10 0 10 2
2 2
10 3 10 3 104 10
104 5 10
105 6 10 6 –10 3–10 3
–10 2–10 2 –10 2–10
0 10 0 10 2
2 2
10 3 10 3 104 10
104 5 10
105 6 10 6
–10 2–10 2 –10 2–10
0 10 0 10 2
2 2
10 3 10 3 104 10
104 5 10
105 6 10 6 –10 2–10 2 –10 2–10
0 10 0 10 2
2 2
10 3 10 3 104 10
104 5 10
105 6 10 6
GFPGFP
fluorescence
fluorescence
(BL1(BL1
510/10)
510/10) GFPGFP
fluorescence
fluorescence
(BL1(BL1
510/10)
510/10)
GFPGFP
fluorescence
fluorescence
(BL1(BL1
510/10)
510/10) GFPGFP
fluorescence
fluorescence
(BL1(BL1
510/10)
510/10)

10 6 10 6 10 6 10 6
540/30)
540/30)

540/30)
540/30)
C 10 6
10 5
10 6
10 5
D 10 6
10 5
10 6
10 5
540/30)
540/30)

540/30)
540/30)

10 5 10 5 10 5 10 5
104 104 104 104
(BL2
(BL2

(BL2
(BL2

104 104 104 104

(BL2
(BL2

(BL2
(BL2

10 3 10 3 10 3 10 3
10 3 10 3 10 3 10 3
fluorescence
fluorescence

fluorescence
fluorescence
fluorescence
fluorescence

fluorescence
fluorescence

10 2 10 2 10 2 10 2
1002 1002 1002 1002
–1002–1002 –1002–1002
–10 2–10 2 –10 2–10 2
YFP
YFP

YFP
YFP
YFP
YFP

YFP
YFP

–10 3–10 3 –10 3–10 3

–10 3–10 3
–10 2–10 2 –10 –10
2
0 100 10
2 2 2
10 10 10 10
3 3 4
10 10
45
10 10
56 6 –10 3–10 3
–10 2–10 2 –10 2–10
0 10 0 10 2
2 2
10 3 10 3 104 10
104 5 10
105 6 10 6
–10 2–10 2 –10 2–10
0 10 0 10 2
2 2
10 3 10 3 104 10
104 5 10
105 6 10 6 –10 2–10 2 –10 2–10
0 10 0 10 2
2 2
10 3 10 3 104 10
104 5 10
105 6 10 6
GFPGFP
fluorescence
fluorescence
(BL1(BL1
510/10)
510/10) GFPGFP
fluorescence
fluorescence
(BL1(BL1
510/10)
510/10)
GFPGFP
fluorescence
fluorescence
(BL1(BL1
510/10)
510/10) GFPGFP
fluorescence
fluorescence
(BL1(BL1
510/10)
510/10)

Figure 25. Flow cytometric detection of dual expression of GFP and YFP. (A) Shows untransfected cells. U2OS cells were transfected with
vectors encoding GFP or YFP, either (B, C) individually or in (D) combination. Samples were acquired and analyzed using the Attune NxT Flow
Cytometer at a flow rate of 200 μL/min. A total of 400,000 cells were collected for the sample coexpressing both fluorescent proteins, and a minimum
of 5,000 events were collected for each control sample. The 488 nm laser was used for excitation of both fluorescent proteins. Coexpression of GFP
and YFP is shown in the upper-right quadrant of (D), and the lower-right quadrant shows cells expressing only GFP.

A A B B
A B

Figure 26. Use of the Attune NxT Fluorescent Protein Filter Kit. The standard configuration for the 561 nm yellow and 488 nm blue laser optical
filter blocks is shown in (A), and the same optical filter blocks using the Attune NxT Fluorescent Protein Filter Kit are shown in (B), with changes
outlined in red.

25
Microbiology research solutions
Bacterial analysis

The Attune NxT Flow Cytometer enables well-separated

bacterial populations. For researchers working with Yellow laser (561 nm) excitation
E. coli, the available green 532 nm laser delivers distinct
A 106
separation of double-live populations of dividing cells Dead
(green) and dead (red) E. coli cells (Figures 27 and 28).

Benefits Live

PI 561 nm 620/15
105
• Easily, reliably, and quantitatively distinguish live and
dead bacteria in minutes
• Handle bacteria samples with improved
clogging resistance 104

• Run dilute samples in minutes

104 105

Data SYTO 9 488 nm 530/30

106
B 105

Dead
Live
PI 532 nm 620/15

105
Live
PI 488 nm 695/40

105

104
Dead
104
103

104 105
104 105
SYTO 9 488 nm 530/30 SYTO 9 488 nm 530/30

Figure 27. Staining of E. coli cells using the Invitrogen™ BacLight™ Figure 28. Staining of E. coli cells using the BacLight LIVE/DEAD
LIVE/DEAD Bacterial Viability Kit and blue laser excitation of Bacterial Viability Kit using either 561 nm or 532 nm excitation of PI.
Invitrogen™ SYTO™ 9 dye and propidium iodide (PI). Samples were E. coli cells were grown in lysogeny broth (LB) and harvested. Samples
analyzed on the Attune NxT Flow Cytometer at the 12.5 μL/min flow rate were analyzed on the Attune NxT Flow Cytometer at the 12.5 μL/min flow
with an event rate of approximately 5,000 events/second. Instrument rate with an event rate of approximately 5,000 events/second using the
settings (voltages, threshold, and advanced settings) were set using blue 488 nm laser and 530/30 nm emission (BL1) for SYTO 9 detection,
single-color controls. The blue 488 nm laser was used for fluorescence and (A) yellow 561 nm laser 620/15 nm emission (YL2) for detection of
excitation of both SYTO 9 dye and PI. SYTO 9 fluorescence emission was propidium iodide (PI). (B) Green 532 nm laser 620/15 nm emission (GL2)
collected using a 530/30 nm emission (BL1), whereas propidium iodide was used for detection of PI. BL1 and SSC thresholds were set and
fluorescence emission was collected using the 695/40 nm emission no compensation was performed. The live population of dividing cells
(BL2). In this example, a BL1 and SSC threshold was used, and results (Live) is shown in green; dead cells (Dead) are shown in red. Excitation
are shown without compensation. The live, SYTO 9–positive population of SYTO 9 and PI with different lasers results in better separation of
is shown in green, and dead cells (Dead) are shown in red. Live and dead the populations.
cell populations are easily distinguished.

26
Oncology research solutions
13-color human lymphocyte immunophenotyping panel

Flow cytometry is the method of choice for identifying cells Benefits

within complex populations, as it allows for multiparameter • Easier design of multicolor panels—improve choices
analysis of thousands to millions of cells in a short time. of reagents
Lymphocyte, monocyte, and granulocyte populations • Excellent cell population resolution for 13-color human
were distinguished with FSC and SSC; and monocyte, lymphocyte immunophenotyping experiments
T cell, B cell, and natural killer (NK) cell populations were
• Strong signal separation for more data clarity
identified using fluorescently labeled antibodies against
surface antigens specific for the different immunological
populations (Figure 29).

Data
A B C D
Side scatter (10 3)

Side scatter (10 3)

Side scatter (10 3)

CD14 Qdot 705

E Propidium Iodide F CD45 Pacific Orange G Forward scatter (10 3) H CD33 PE Cy7

CD62LAPC Alexa Fluor 750

CD4 PerCP Cy5.5
CD19 Pacific Green
CD45RA FITC

HLA-DR PE Cy7 CD3 Alexa Fluor 700 CD8 Pacific Blue CD45RA FITC
I J K
CD8 Pacific Blue

CD25 APC

CD56 PE HLA-DR PE Cy7 CD45RA FITC

Figure 29. Gating strategy. (A) Dead cells were excluded from the analysis by gating on live cells in a dot plot. (B) CD45+ cells were gated on
to select the leukocyte population from the lysed whole blood. (C) Lymphocytes and monocytes were gated based on forward and side scatter
profiles. (D) Monocytes are found above the lymphocytes based on scatter profiles and express both CD14 and CD33. (E) B cells can be further
characterized by HLA-DR and CD45RA expression. (F) Within the lymphocyte gate, T cells can be isolated based on their expression of CD3 and
(G) further subdivided into CD4 (T helper cell) and CD8 (cytotoxic T cell) subpopulations. (J) In addition, regulatory T cells express CD4 and CD25.
(H and K) CD62L identifies naïve (TN) CD4 and CD8 T cells, whereas HLA-DR is expressed by activated T cells (TA). (I) NK cells can be identified as
they lack B cell (CD19) and T cell (CD3) markers, and express CD56.

27
Immuno-oncology research solutions

Innate lymphoid cells (ILCs) are rare populations of

cytokine-producing lymphocytes that express no unique
cell surface markers. ILCs can however be identified by
combinations of multiple cell surface markers, making flow
cytometry the best method for their detection. 

Benefits
• Run large sample volumes in a fraction of the time for
rare-cell detection
• No need to concentrate your sample
• Achieve a reliable measure of accuracy for detection of
cell populations comprising less than 1% of the total cells
by easily collecting millions of events (Figure 30)

Data

A 1.0M B 1.0M C 10 6

10 5
800K 800K

10 4
Lineage

600K 600K
SSC-A
FSC-H

Lymphocytes
Singlets 66.9
400K 59.7 400K 10 3

200K 200K 10 2
I.C2
0 0016
0 0
0 200K 400K 600K 800K 1.0M 0 200K 400K 600K 800K 1.0M 0 10 3 10 4 10 5 10 6

FSC-A FSC-A CRTH2

Figure 30. Detection of rare ILC2 population in PBMCs. (A) Labeling of 1 x 10 6 PBMCs resuspended in 100 µL PBS (+10% FBS). The antibodies
used were a lineage cocktail containing CD2, CD3, CD14, CD16, CD19, CD56, and CD235a conjugated to Invitrogen™ FITC, CD123-FITC, and
CRTH2-Alexa Fluor™ 647 conjugates. The ILC2 cells are then defined as the lineage (BL1)-negative, CRTh2 (RL1)-positive populations. (B) CRTH2 cells
expressing the chemoattractant receptor–homologous molecule expressed on Th2 cells. CRTH2, is a seven-transmembrane protein coupled with
heterotrimeric G proteins. CRTH2 is the prostaglandin D2 receptor and is expressed by Th2 cells, eosinophils, and basophils. CD294 prevents the
apoptosis of Th2 cells and mediates the chemotaxis of CRTH2-expressing cells to the sites of allergic inflammation, such as the asthmatic lung. (C)
The ILC2 cells are defined as lineage-negative and CRTH2-positive. In this example, the ILC2 population is 0.016% of the parent gate. Data courtesy
David Cousins, University of Leicester.

28
Flow cytometry reagents

Enable and explore a bright, expanded world of flow Reagents—At the forefront of invention and development
cytometry with Invitrogen™ fluorescence detection of fluorescent probes for over 40 years, we offer a
molecules and probes, which are backed by 40 years comprehensive variety of cell functional assays for studying
of pioneering R&D. From conjugated antibodies through viability, apoptosis, cell cycle, and cell proliferation.
functional dyes and cell functional assays, our flow
cytometry products exist to pioneer your research. Flow support products—Compensation beads are
essential to perform quantitative measurements on
Go to thermofisher.com/flow-cytometry for more individual cells and other particles with high precision,
information on Invitrogen flow cytometry products speed, and accuracy, especially when performing flow
and resources. cytometry using multiple channels, markers that are
poorly expressed, or from limited sample. As with all
Accelerate your science with a comprehensive suite of high-performance instrumentation, flow cytometers
solutions for the analysis of cells and their function with must also be calibrated regularly to ensure accuracy and
Invitrogen™ eBioscience™ flow cytometry antibodies and reliability. The stability, uniformity, and reproducibility of
Invitrogen™ cell health reagents. Invitrogen™ microsphere products make them excellent
tools for flow cytometer instrument setup and calibration.
Antibodies—Build and expand your panels using over
15,000 flow-specific conjugated antibodies with multiple We are focused on advancing meaningful discoveries
fluorophore options, including the new Super Bright and partnering to make tools for cellular analysis widely
violet-excitable polymer dyes. accessible, affordable, and powerful for all life scientists.
On the quest for significant breakthroughs, we know that
Buffers—The use of appropriate buffers is crucial to the you never settle for average, and neither will we.
success of your flow cytometry experiments. We offer a
wide variety of buffers to suit your research needs, whether
your experiment calls for extracellular, intracellular, and/or
nuclear cell staining.

29
Robotic automation solutions
Orbitor RS Microplate Mover

Maximize operating capacity, mitigate human operator

Extended
error, and enable rich, reproducible data with the
Thermo Scientific™ Orbitor™ RS Microplate Mover as part of
a comprehensive, multicomponent workcell for robotically
10 L
fluidics
automated flow cytometry.

Technology Available
The robotic arm offers active and passive protective safety
features, demonstrated reliability, and flexible configuration
4–40˚C
storage
options for arrangement and storage. Operation is
managed by Thermo Scientific™ Momentum Scheduling
Software, established with instrument drivers available for Up to
over 200 instruments. The dashboard facilitates dynamic
scheduling for active prioritization, visualized progress, 19.5 hours
and plate tracing. Extended-run fluidics allow for up to continuous runtime*
19.5 hours of unattended continuous runtime under
specific run conditions.

Benefit
• Robust performance, precise motion, and
consistent performance
• Compatible with a diverse range of plate types
• Works with both lidded and unlidded plates

The Attune NxT Flow Cytometer configured for robotic automation with the Orbitor RS Microplate Mover.

* Under specific run conditions.

30
Walk away with confidence
Labs optimized with robotic handling will benefit from the • Mitigate evaporation—the Orbitor RS Microplate Mover
performance, software design, engineering, and safety can de-lid and re-lid plates as they are loaded, unloaded,
features of the Orbitor RS Microplate Mover. and stored

• Temperature sensitivity—optional, temperature- • Protect from light exposure—opaque and lidded

controlled Thermo Scientific™ SmartStor™ benchtop plates protect samples in the random access hotel
microplate storage device with a temperature range of storage tower
4–40°C
Find out more at thermofisher.com/flowautomation
• Flexible capacity—plate capacity of 20 standard
microplates or 9 deep-well blocks, and self-scanning
internal inventory

31
Transforming capabilities
Options that resonate

With additional lasers, more detection channels, increased flexibility, and

design modifications that further improve the performance, reliability, and
robustness, the Attune NxT Flow Cytometer continues to offer more options
and added functionality. Since its initial unveiling, the compact system with
innovative acoustic technology is moving ahead with added new functionalities
and capabilities.

“I knew I had learned a lot during 25 years of

experience doing research with flow cytometry. Now
I am surprised to see how much I can learn doing
research with the Attune NxT Flow Cytometer, and how
this new technology can be very helpful to make the
invisible visible.”

– Jordi Petriz, PhD

José Carreras Leukaemia Foundation

32
Timeline—stay tuned for what’s next

2014 2016 2018

Invitrogen™ Attune™ NxT
Flow Cytometer introduced
Invitrogen™ Attune™ NxT
Green laser (532 nm) launched
Preview Thermo Scientfic™
Orbitor™ RS Microplate Mover

2015 2017
Invitrogen™ Attune™ NxT Software
support for deep-well plates
Invitrogen™ Attune™ NxT
(Violet 6-channel configuration)
(2 mL) and sample recovery

Invitrogen™ Attune™ NxT

No-Wash No-Lyse Filter Kit
Invitrogen™ Attune™ NxT
Fluorescent Protein Filter Kit

33
Aftermarket care
Partner with a flow cytometry company invested
in supporting you through a lifetime of research
Choose a service plan that is right for you—beyond repair to
proactive care

• Peace of mind—during every stage of ownership: instrument install, repair,

and maintenance
• Flexible service options—over 1,000 technical specialists delivering 30 years
of experience servicing life sciences instrumentation
• AB Assurance plan and extended warranty—covers all costs associated
with instrument repairs

Ordering information
Product* Description Cat. No.
AB Maintenance including
Attune NxT 1-Laser System ZG51SCATTUNEB
1 planned maintenance (PM)
Attune NxT 1-Laser System AB Assurance including 1 PM ZG11SCATTUNEB
Attune NxT 2-Laser System AB Maintenance including 1 PM ZG51SCATTUNEBRBVBY
Attune NxT 2-Laser System AB Assurance including 1 PM ZG11SCATTUNEBRBVBY
Attune NxT 3-Laser System AB Maintenance including 1 PM ZG51SCATTUNEBRVBVY
Attune NxT 3-Laser System AB Assurance including 1 PM ZG11SCATTUNEBRVBVY
Attune NxT 4-Laser System AB Maintenance including 1 PM ZG51SCATTUNEBVRY
Attune NxT 4-Laser System AB Assurance including 1 PM ZG11SCATTUNEBVRY
Attune Operation Qualification and Instrument
Attune IQ/IPV 4465413
Performance Qualification (IQ/IPV)
Attune Installation Qualification and
Attune IQ/OQ 4465445
Operation Qualification (IQ/OQ)
Orbitor RS AB Protection Orbitor Robot NxT ZG30SCORBROBNXT

* Instrument needs to be networked.

34
Ordering information
Unit type Configuration Parameter Cat. No.
Blue/red/yellow/violet A24858
4-laser Blue/red/violet/green 16 A29001
Blue/red/yellow/violet 6 A29004
Blue/red/violet 6 14 A29003
Blue/red/violet A24860
13
Blue/violet/yellow A24859
3-laser
Blue/red/yellow 12 A28993
Blue/green/violet 13 A28999
Blue/green/red 12 A28997
Blue/violet 6 11 A29002
Blue/violet 10 A24862
2-laser Blue/red A24863
Blue/yellow 9 A24861
Blue/green A28995
1-laser Blue 6 A24864

35
Ordering information
Product Cat. No.
Attune NxT accessories
Attune NxT Autosampler 4473928
Attune NxT External Fluid Supply A28006
Attune NxT Software, Single License A25554
Attune NxT Software, 5 Licenses A24856
Attune NxT Software, 10 Licenses A24855
Orbitor RS Microplate Mover Stack A33007
Orbitor RS Microplate Mover Hotel A33008
Orbitor RS Microplate Mover Stack/Hotel A35220
Attune NxT upgrades
Attune NxT Yellow Laser Upgrade Kit 100022779
Attune NxT Red Laser Upgrade Kit 100022778
Attune NxT Green Laser Upgrade Kit A32701
Attune NxT Violet 6 Conversion Kit, Blue Laser A35428
Attune NxT Violet 6 Conversion Kit, Violet Laser A36569
Attune NxT Violet 6 Conversion Kit, Red Laser A36571
Attune NxT Violet 6 Conversion Kit, Yellow Laser A36572
Attune NxT Fluorescent Protein Filter Kit—GFP, YFP, mCherry 100022775
Attune NxT No-Wash No-Lyse Filter Kit 100022776
Attune NxT Custom Filter Holder Kit A27784
Attune NxT reagents and consumables
Attune Debubble Solution (1X), 50 mL A10496
Attune Focusing Fluid (1X), 1 L 4488621
Attune Focusing Fluid (1X), 10 L A24904
Attune Wash Solution, 250 mL A24974
Attune Shutdown Solution (1X), 250 mL A24975
Attune Performance Tracking Beads 4449754

Find out more at thermofisher.com/attune

For Research Use Only. Not for use in diagnostic procedures. Not for resale. Super Bright Polymer Dyes are sold under
license from Becton, Dickinson and Company. © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are
the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. BD and BD Horizon are trademarks of
Becton, Dickinson and Company. PE/Dazzle is a trademark of BioLegend Inc. VioBlue and VioGreen are trademarks of Miltenyi
Biotec. Brilliant Violet is a trademark of BD Biosciences. COL09360 0419