Sample Data for the Attune NxT Flow Cytometer

Designed using acoustic-assisted hydrodynamic focusing, theAttune 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).

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.

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.

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.

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).

• See application note: Attune NxT Flow Cytometer for 6-color immunophenotyping analysis of stained human whole blood using a no-lyse, no-wash protocol, with no compensation 
• Read the BioProbes 71 article: No-Compensation Immunophenotyping Using a Four-Laser Flow Cytometer

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).

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.

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).

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.

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).

• Read the BioProbes 71 article: Tools and strategies for rare-event detection using flow cytometry
• See application note: Detection of platelets in whole blood using the Attune NxT Flow Cytometer 

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.

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).

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.

• See application note: Multiparameter analysis of murine regulatory T cells and dendritic cells with the Attune NxT Flow Cytometer
• See application note: Ten-parameter immunophenotyping of human lysed whole blood with the Attune NxT Flow Cytometer allows for the identification of multiple T cell subsets and myeloid cells
• View all of the Attune NxT Flow Cytometer application notes

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).

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.

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.

• Download application note: Detection of fluorescent proteins using the Attune NxT Flow Cytometer
• Read the BioProbes 71 article: Multicolor detection of fluorescent protein expression with the Attune NxT Flow Cytometer

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).

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.

• See the application note: Flow cytometry analysis of transcription factor expression during differentiation of hPSC-derived cardiomyocytes

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