styled illustration of flow cytometer results

Enabling your more challenging experiments

Equipped with acoustic-assisted hydrodynamic focusing and fluidics designed to minimize clogging and effectively handle a broad range of cell types and samples, the Attune NxT Flow Cytometer helps you get more—more data, more detail, and more throughput.

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Learn more about the benefits of acoustic focusing

Performance data

Efficient—10X faster speed with no loss in data quality

Designed using acoustic-assisted hydrodynamic focusing, the Attune NxT Flow Cytometer achieves sample-throughput rates of 12.5–1 mL/min—up to 10 times faster than traditional hydrodynamic focusing systems—and data acquisition speeds of 35,000 events/sec. This means that you can process all of your samples—including low-concentration and precious samples—more quickly and accurately than ever before with no loss in quality (Figure 1).

4 panel bar graphs comparing speed of Attune NxT Cytometer to competitor instruments
table showing similar CV values at six different sample rates

Figure 1. Attune NxT Flow Cytometer processes samples in a fraction of the time required for three competitor instruments with no loss in data quality. (Top) Times taken to acquire 10,000, 100,000, 1,000,000, and 10,000,000 events, based on high (1,000 µL/min) flow rate in the Attune NxT Flow Cytometer and maximum flow rates for three competitor instruments were calculated and plotted. Calculations assume sample concentration of 106 cells/mL and the following flow rates (obtained from competitor marketing materials): Competitor A: 66 µL/min. Competitor B: 120 µL/min. Competitor C: 60 µL/min. (Bottom) Minimal data variation at high sample rates. Jurkat cells were fixed and stained with propidium iodide, treated with RNase and analyzed at a concentration of 1 x 106 cells/mL on the Attune NxT Flow Cytometer. Regardless of sample rate, the coefficient of variation (CV) of cells in the G0/G1 and G2/M phases remain consistent, even at the highest sample rate of 1,000 μL/min.


Consistent—Minimal data variation regardless of sample rate

Cell cycle analysis is just one example of where it is critical to precisely detect differences in fluorescence intensity between multiple cell populations. With the Attune NxT Flow Cytometer, minimal variation in results is seen regardless of sample throughput rate (Figure 2). You no longer need to sacrifice throughput for sensitivity.

6 panels of histograms showing minimal data variation at high sample rates with the Attune  NxT Acoustic Focusing Cytometer

Figure 2. Minimal data variation at high sample rates with the Attune NxT Flow Cytometer. Jurkat cells were alcohol-fixed and stained with propidium iodide, treated with RNase, and analyzed at a concentration of 1 x 106 cells/mL on the Attune NxT Flow Cytometer at different sample rates. The left peak in all graphs reflects cells in G0/G1 phase, while the right peak reflects cells in G2/M phase. Regardless of sample rate, the widths of the G0/G1 and G2/M peaks and CV% remain consistent for the Attune NxT Flow Cytometer, even at the highest sample rate of 1,000 µL/min.

Flexible—Minimal compensation panels

Compensation is not simple, requiring runs of positive and negative, color-matched controls in conjunction with careful monitoring of background fluorescence. The Attune NxT Flow Cytometer can be configured with up to 4 spatially separated lasers, giving you the flexibility to build multicolor panels that are well separated spectrally and do not have significant overlap, requiring minimal compensation (Figure 3).

5-panel histogram figure showing optimal design of a no-lyse/no-wash, 6-color immunophenotyping panel for human T cell subsets
Click image to enlarge

Figure 3. Optimal design of a no-lyse/no-wash, 6-color immunophenotyping panel for human T cell subsets acquired on the Attune NxT Flow Cytometer without using compensation at any step. Human whole blood was stained with 6 probes and analyzed on the Attune NxT Flow Cytometer. (A) A fluorescence threshold was set on Pacific Orange fluorescence (CD45), and events coincident with red blood cells were excluded based on PE positivity (glycophorin A or Gly A). (B) Lymphocytes were gated based on scatter properties, from which (C) T cells were identified by CD3 expression. (D, E) T cells were then analyzed for their expression of the lineage markers CD4 and CD8 as well as the activation marker CD62L in order to identify naive/central memory T cells (CD62L-positive) and effector memory T cells (CD62L-negative). No compensation was required to analyze or display these data.


Transformative—Precision and sensitivity at all sample rates

The Attune NxT Flow Cytometer enables higher sensitivity to better distinguish between dim signals and background resulting in less variation and better signal separation (Figure 4).

3 panel figure of histograms showing sensitivity measurements across flow rates

Figure 4. Sensitivity measurements across flow rates. Fluorescent microspheres (Spherotech Rainbow 3.2 μm) were run on a high-end conventional flow cytometer (A) and on the Attune NxT Flow Cytometer (B and C) using a 561 nm laser and 610/20 (A) or 610/15 (B and C) emission filters. The conventional cytometer was run using the highest sensitivity setting (~12.5 μL/min). The Attune NxT Flow Cytometer was run at 12.5 μL/min (B), which is equivalent to the traditional flow cytometer and 500 μL/min (C; 40x more sample). The Attune NxT Flow Cytometer results showed equal or better results even at the highest flow rates.

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Immuno-oncology

Achieve a reliable measure of accuracy for rare events

With the Attune NxT Flow Cytometer you can achieve a reliable measure of accuracy for detection of cell populations comprising less than 1% of the total cells by easily running large sample volumes in a fraction of the time without the need to concentrate your sample (Figure 5).

Detection of rare ILC2 population in PBMCs

Figure 5. Detection of rare ILC2 population in PBMCs. (A) Labeling of 1 x 106 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.


Run large sample volumes in a fraction of the time

Detection of rare events includes populations of cells comprising less than 1% of total cells, which includes the detection of stem cells, minimal residual disease cells, natural killer cells and fetomaternal hemorrhage cells. The Attune NxT Flow Cytometer allows dilute samples to be processed quickly at sample input speeds of up to 1 mL/min (Figure 6).

Mouse plasmacytoid dendritic cell (pDC) gating and analysis

Figure 6. Collecting more than 1 million live cells and detecting a rare population of dendritic cells of 0.2% with mouse splenocytes. Plasmacytoid dendritic cells (pDCs) are a specialized cell population that produces large amounts of type I IFNs in response to viruses and are identified using the immunophenotype CD19–/B220high/CD317+. Four-color staining of mouse splenocytes included CD19-Pacific Blue, CD317-Alexa Fluor 488, CD45R/B220-PE direct conjugates, and SYTOX AADvanced Dead Cell Stain. A gate was made on live cells using SYTOX AADvanced Dead Cell Stain, followed by gating on CD19– cells. A two-parameter plot of CD45R/B220 vs. CD317 was used to identify pDCs. A collection rate of 500 μL/min was used to acquire 1.3 million total cells with a cell concentration of 7.5 x 107 cells/mL. Plasmacytoid dendritic cells were identified as dual B220+/CD317+ (upper right quadrant) and constitute 0.851% of live CD19– cells, which is 0.194% of total splenocytes.

Thermo Fisher Scientific offers many research platforms and products to help you better understand the interplay between the immune system and cancer. Explore our immuno-oncology solutions at thermofisher.com/immunooncology.

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Immunophenotyping

Easier design of multicolor immunophenotyping panels

With the option to be configured with up to 4 lasers and 14 colors for multiparameter analysis, the Attune NxT Flow Cytometer can be designed to accommodate the most common fluorophores used in flow cytometry to match the immunophenotyping  panels you are currently running (Figure 7).

Multiparameter (10-color) analysis of cells with the Attune® NxT Acoustic Focusing Cytometer
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Figure 7. Multiparameter (10-color) analysis of murine regulatory T cells and dendritic cells with the Attune NxT Flow Cytometer. Lymphocytes were gated using FSC/SSC parameters (A, left) and B220-expressing B cells were omitted from subsequent analysis (A, middle). Within the B220–, CD45.2+ gate, T cells were analyzed based on their expression of CD3 (A, right). CD3+ T cells were separated into two populations based on expression of the co-receptors CD4 or CD8 (B, left). Within the CD4+ T cells there is a subpopulation of suppressive regulatory T cells that express the transcription factor Foxp3 and the cell surface marker CD25 (IL-2Rα) (B, right). CD3– cells were separated to show a rare population of CD11c+ MHCII+, professional antigen-presenting dendritic cells (C, left). Splenic dendritic cells can be subdivided further into CD11b+ and CD8+ dendritic cell subsets (C, right), each possessing unique antigen presentation properties.

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Microbiology

Quick and accurate detection of wastewater

On traditional flow cytometers, very dilute samples can take a long time to acquire due to slower flow rates. The Attune NxT Flow Cytometer can run very dilute samples quickly (Figure 9).

Analysis of bacteria in treated municipal wastewater on the Attune NxT Flow Cytometer

Figure 9. Analysis of bacteria in treated municipal wastewater on the Attune NxT Flow Cytometer. A 3 mL sample of municipal wastewater was labeled with the Invitrogen LIVE/DEAD BacLight Bacterial Viability Kit and analyzed on the Attune NxT Flow Cytometer at a flow rate of 1 mL/min, which allowed quick analysis of the sample and accurate detection of very small quantities of bacteria. Concentrations of the live and dead bacteria where determined without using reference counting beads. The two-parameter dot plot (propidium iodide vs. SYTO 9 fluorescence), with the live (green) and dead (red) bacterial populations are well separated; the statistics table displays the concentration measurements for the labeled bacteria. Wastewater may also include small eukaryotes and types of bacteria that are potentially viable but nonculturable, each of which may also be labeled with the dyes; the grey dots represent debris found in the wastewater.

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Fluorescent proteins

Detect multiple fluorescent proteins

The Attune NxT Flow Cytometer has a modular design with a 488 nm laser for excitation of the most commonly used FP (EGFP) and its variants (emGFP, TurboGFP), and can be upgraded to include optional laser lines including 405 nm, 561 nm, and 637 nm lasers. The 561 nm laser is particularly useful for exciting the orange- and red-fluorescent protein variants. The 405 nm laser can be used to excite FPs such as TagBFP, the bright blue-fluorescent mutant created from site-specific and random mutagenesis of TagRFP, or others including Azurite and T-Sapphire. Figure 10 shows detection of TagBFP, emGFP, YFP, mOrange2, TagRFP, mKate, and mCherry using the Attune NxT Flow Cytometer.

Histograms showing detection of a palette of fluorescent proteins using the Attune NxT Flow Cytometer
Click image to enlarge

Figure 10. Detection of a palette of fluorescent proteins using the Attune NxT Flow Cytometer. 293FT cells or U2OS cells were transfected or transduced with plasmid or viral constructs expressing different fluorescent proteins. Samples were acquired on the Attune NxT cytometer at a flow rate of 100 μL/min using 405 nm, 488 nm, or 561 nm excitation sources. (A) Blue Fluorescent Protein (TagBPF) fluorescence was collected in the VL1 channel using a 440/50 bandpass (BP) filter; (B) Emerald GFP (emGFP) fluorescence and (C) Yellow Fluorescent Protein (YFP, Venus variant) fluorescence (in cells transduced with the Premo Halide Sensor) were collected in the BL1 channel using a 530/30 BP filter; (D) Orange Fluorescent Protein (mOrange2) fluorescence and (E) Red Fluorescent Protein (TagRFP) fluorescence were collected in the YL1 channel using a 585/16 BP filter; (F) mKate fluorescence and (G) mCherry fluorescence were collected in the YL2 channel using a 620/15 BP filter. Control cells that do not express fluorescent protein are shown in each histogram overlay (gray peaks). TagBFP, mOrange2, TagRFP, YFP, and mCherry were expressed from the CMV promoter, and emGFP and mKate were expressed from the EF-1α promoter. YFP and RFP constructs were delivered to U2OS cells using the BacMam 2.0 transduction system, whereas TagBFP, emGFP, mKate, and mOrange2 constructs were transfected into 293FT cells using Lipofectamine 3000 reagent. The mCherry construct was transduced into U2OS cells using an adenovirus delivery system.


Quickly monitor transfection efficiencies

Flow cytometry fits into the CRISPR analysis workflow, enabling researchers to monitor the efficiency of genome editing experiments. When screening libraries or large sample populations of edited cells, the Attune NxT Flow Cytometer system enables razor-precision analysis. With appropriate antibodies, fluorescent proteins, or functional probes, complex phenotypes can be unraveled through multiplexing (Figure 11).

tracking-blue-fluorescent-protein

Figure 11. Tracking blue fluorescent protein (BFP) converting to green fluorescent protein (GFP) by homologous recombination using the CRISPR-Cas9 system. Single-stranded oligodeoxynucleotides (ssODN) assist in making large genomic changes following cleavage by Cas9 nuclease and in vitro–transcribed guide RNA (IVT gRNA).

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Cell types

Gentle and safe analysis for stem cells

The ability to direct human pluripotent stem cells (hPSCs) toward differentiated cell phenotypes offers tremendous potential for personalized and regenerative medicine. The Attune NxT Flow Cytometer is ideally suited for use with fragile and large cell types like stem cells and cardiomyocytes (Figure 13). Engineered to actively resist clogging, a syringe-driven system and larger flow cell help prevent the loss of precious sample and is drastically less susceptible to clogs.

Flow cytometry analysis of transcription factors during cardiomyocyte differentiation
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Figure 13. Flow cytometry analysis of transcription factors during cardiomyocyte differentiation. 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.

Table 1. Cell types successfully analyzed on the Attune NxT Flow Cytometer.

Sample type Demonstrated example Published References
Algae Nannochloropsis oculata  
Bacteria Escherichia coli
Bacillus subtilis
Legionella strains
Staphyloccus aureus  
Diatom Phaeodactylum tricornutum
Human Human adenocarcinoma
Human bone marrow (PBMCs)
Human bone osteosarcoma cells (U2OS)
Human cardiomyocytes  
Human corneal epithelial cells  
Human embryonic kidney (293FT)
Human endothelial cells
Human leukocytes  
Human mesenchymal stem cell (hMSC)
Human red blood cells (RBC)  
Human T cell leukemia (Jurkat)
Human whole blood
Mouse Mouse splenocytes
Mouse T and B regulatory cells
Mouse cardiomyocytes
Mouse bone marrow
Mouse lung tissue
Mouse tumor cells
Mouse skeletal muscle
Parasites Plasmodium falciparum
L. amazonensis, T. brucei and T. cruzi
Trypanosoma cruzi
Picophytoplankton Prochlorococcus spp;
Synechococcus spp
 
Plants Arabidopsis thaliana  
Phalaris arundinacea
Solidago canadensis
Yeast Candida albicans
Saccharomyces cerevisiae
Zebra fish Zebra fish organs
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