The Attune® NxT Acoustic Focusing Cytometer

Introduced in 2010, the first-generation Attune® cytometer revolutionized flow cytometry by using acoustic focusing to precisely align cells, breaking away from traditional cell alignment by hydrodynamic focusing. Here we announce the arrival of the next generation in acoustic focusing cytometry (Figure 1). The Attune® NxT Acoustic Focusing Cytometer retains all the focusing benefits of its predecessor, including rapid processing of dilute samples and sample input flow rates that span two decades of dynamic range. Moreover, it expands on its first-generation legacy with additional lasers, more detection channels, increased flexibility, and design modifications that further improve the performance, reliability, and robustness of the instrument (Table 1).

Photograph of the Attune® NxT Acoustic Focusing Cytometer and the Attune® NxT Autosampler  

 

Figure 1. The Attune® NxT Acoustic Focusing Cytometer with optional Attune® NxT Autosampler, which enables processing of up to 384 samples.

Table 1. Comparison of first-generation Attune® cytometer with second-generation Attune® NxT cytometer.

Feature Attune® cytometer
(first generation)
Attune® NxT cytometer
Number of lasers 2 Up to 4
Number of detection channels 8 Up to 16
Sample flow rates (µL/min) 25–1,000 12.5–1,000
Detection speed (events/sec)
10,000 35,000
Size (H x W x D) 40 x 58 x 43 cm
16 x 23 x 17 in
40 x 58 x 43 cm
16 x 23 x 17 in

Small in size but big in performance

Customer feedback on the first-generation Attune® cytometer highlighted the need for additional laser and color options to meet the growing demand for multicolor analyses. The Attune® NxT cytometer was therefore designed with more laser and detection channel choices while keeping the same benchtop size of the original 2-laser Attune® system. With a footprint similar to that of a microwave oven, the Attune® NxT can be configured with up to 4 spatially separated lasers (405 nm, 488 nm, 561 nm, and 637 nm) and 16 detection channels. Figure 2 shows the multiparameter (10-color) analysis of murine regulatory T cells and dendritic cells with the Attune® NxT Acoustic Focusing Cytometer. Panel design is critical in multicolor experiments; to learn more about designing an effective multiplex panel, see the description of the webinar “Basics of multicolor flow cytometry panel design”.

This new design not only accommodates the most common fluorophores and fluorescent proteins used in flow cytometry but also reduces the need for compensation by utilizing spatially separated laser technologies. The unique modular design of the Attune® NxT cytometer enables up to 7 different laser configurations ranging from a 1-laser/6–detection channels system to a 4-laser/16–detection channels system. Researchers can acquire additional lasers and detection channels over time with simple upgrade packages that can be executed in their labs, allowing the utility of this instrument to grow and expand with the demands of the experiments.

7 panel figure of multiparameter (10-color) analysis of murine regulatory T cells and dendritic cells with the Attune® NxT Acoustic Focusing Cytometer

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

Superior reliability with maximal signal efficiency

Instrument downtime is a large concern with many flow cytometry users, and laser alignment is often the underlying cause of this work interruption. Most flow cytometers use lasers that have a Gaussian profile on the flow cell, demanding that laser alignment be very precise. In contrast, the Attune® NxT cytometer is equipped with flat-top lasers; their flat intensity profile allows a much wider window of alignment. This novel optical design helps ensure precise fixed alignment of all lasers onto the sample stream even at high sample rates, leading to consistency in data over time and first-class reliability. State-of-the-art fiber-optic cables are used to deliver laser light to the flow cell, as well as to transport light emitted from the cells to the detection optics, adding to the stability of the instrument.

The Attune® NxT cytometer is designed to provide precision and sensitivity at all sample rates with minimal data variation. Figure 3 shows a cell cycle analysis experiment performed at different sample rates; cell cycle analysis is one example of an application in which it is critical to precisely detect small differences in fluorescence intensity between multiple cell populations. Facilitated by the combination of acoustic and hydrodynamic focusing, the precise sample alignment enables researchers to obtain tight coefficients of variation (CVs) at both low and high sample input rates to better distinguish between dim signals and background, resulting in less variation and better signal separation. Furthermore, the optical detection filters can easily be interchanged and customized to minimize reagent crosstalk and maximize signals.

Five similar histograms showing minimal data variation at high sample rates with the Attune® NxT Acoustic Focusing Cytometer

 
Figure 3. Minimal data variation at high sample rates with the Attune® NxT Acoustic Focusing 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 Acoustic Focusing 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 cytometer, even at the highest sample rate of 1,000 μL/min.

Rapid detection of rare events

A fundamental difference between the Attune® NxT Acoustic Focusing Cytometer and conventional hydrodynamic focusing cytometers is that the focusing of individual cells is largely independent of the sample input rate, allowing cells to be tightly focused regardless of the sample-to-sheath ratio. The Attune® NxT cytometer utilizes acoustic waves in combination with hydrodynamic focusing to align cells for laser interrogation, allowing the researcher to choose hydrodynamic focusing at low sample input rates or a combination of acoustic and hydrodynamic focusing at higher sample input rates. Because there is minimal data variation regardless of sample- throughput rate (Figure 3), the Attune® NxT cytometer is ideal for detecting cell proliferation, cell cycle phase, and rare cell events.

In order to attain accurate measurements, analysis of rare cell populations requires the collection of high numbers of events, which can lead to long acquisition times. Likewise, data from dilute samples like cerebrospinal fluid (CSF), stem cells, and any sample with low cell numbers can take a long time to acquire. With the Attune® NxT cytometer, even dilute samples can be analyzed quickly without compromising data. The Attune® NxT cytometer achieves sample throughput at rates over 10 times faster than other cytometers—up to 1,000 μL/min and 20 million events per run, enabling rapid detection of rare events with precision and accuracy and without aborting data. For example, Professor David Cousins of the University of Leicester reported, “One of the main projects in my laboratory is focused on the description and functional analysis of an emerging innate lymphoid cell type. These cells are extremely rare both in blood and in tissues. For flow cytometry analysis we use the Attune® Acoustic Focusing Cytometer. The high sample rates of the Attune® cytometer allow me to reduce centrifugation steps so that we retain more cells and more rapidly detect rare events. We could not have performed these studies with any other instrument.”

Dilute your samples, not your data quality

The high sample-throughput rates of the Attune® NxT cytometer are opening up new applications, such as no-wash/no-lyse whole blood protocols. Washing and lysing of red blood cells (RBCs) can cause significant cell loss and damage during sample preparation steps required in flow cytometry. Difficult-to-collect samples like mouse blood and bone marrow or thin-needle aspirates can be stained and then diluted without washing or performing RBC lysis.

No-wash/no-lyse protocols minimize cell loss and simplify sample preparation steps by avoiding long and tedious centrifugation steps typically required to process cell samples, and high sample collection rates make faster data acquisition possible—you can run up to 10 mL in just 10 minutes (compared to 100 minutes on a traditional flow cytometer). Furthermore, minimal sample processing and short acquisition times help keep cells healthy, and sample loss is avoided, allowing for full-panel testing with all precious samples. In addition, the Attune® NxT cytometer can easily be configured to customize side scatter from any laser to achieve more flexibility. For example, we have shown that violet side scatter helps differentiate white blood cells from RBCs and can even differentiate lymphocytes from monocytes and granulocytes when combined with blue forward scatter (Figure 4), allowing the identification of three cell types without the use of RBC lysis or antibodies.

Two scatter plots showing that elimination of sample preparation does not compromise data quality with the Attune® NxT Acoustic Focusing Cytometer  
Figure 4. Eliminate sample preparation without compromising data quality. The no-wash/no-lyse protocol was used to analyze normal human whole blood. A 5 μL sample of whole blood was stained with fluorescent antibody conjugates, incubated, then diluted using 4 mL of PBS buffer and analyzed on the Attune® NxT cytometer. The 405 nm light is readily absorbed by red blood cells (RBCs), enabling use of the differential side scatter gating (A), in which a 405 nm vs. 488 nm side scatter dual-parameter plot is used to differentiate the leukocyte and RBC populations. From this gated population, a daughter plot of side scatter vs. forward scatter (B) can be used to identify lymphocyte, monocyte, and granulocyte populations, without the need for a fluorescent label. You can also perform the no-wash/no-lyse method using glycophorin A or CD45 fluorescent antibodies to exclude RBCs using a fluorescence threshold.  

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