Attune® NxT Acoustic Focusing Cytometer
Attune® NxT cytometer software
Changing flow cytometry
With the option to be configured with up to 4 lasers and 14 colors, the Attune® NxT Acoustic Focusing Cytometer was designed as a modular system to fit most experimental design needs and lab budgets. The Attune® NxT cytometer can be designed to accommodate the most common fluorophores and fluorescent proteins used in flow cytometry to match the panels you are currently running. Multiple fluorescent proteins can be detected with an optional 561 nm laser. Whether you choose to design your dream machine now or upgrade later, the Attune® NxT Acoustic Focusing Cytometer can grow with you and your research needs.
Figure 1. Multiparameter (10-color) analysis of murine regulatory T cells and dendritic cells with the Attune® NxT Acoustic Focusing Cytometer. Lymphocytes were gated on 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.
|Figure 2. Live-cell fluorescent protein detection. U2OS cells were simultaneously transduced with CellLight® Nucleus-GFP (Cat. No. C10602) and CellLight® Plasma Membrane-RFP (Cat. No. C10608). Cells were analyzed on the Attune® NxT Acoustic Focusing Cytometer using a 488 nm laser and 530/30 emission filter, and 561 nm laser and 585/16 nm emission filter.|
Table 1. Attune® NxT Acoustic Focusing Cytometer fluorophore selection guide.
|Laser||Excitation||Emission||Common dye||Fluorescent protein|
|Violet||405 nm||440/50||Pacific Blue™||ECFP|
|530/30||FITC||EGFP, Emerald GFP|
|590/40||PE-Alexa Fluor® 610, PE-Texas Red® , PE|
|780/60||Pe-Cy®7, PE-Alexa Fluor® 750, Qdot® 800|
|620/15||PE-Texas Red®||mCherry, dTomato, DsRed, mStrawberry|
|720/30||Alexa Fluor® 700|
|780/60||APC-Alexa Fluor® 750|
|*Without a yellow laser (590/40) suitable for PE.
**Side scatter can be detected by any laser line (default is blue laser). Violet side scatter is recommended for no-wash/no-lyse applications.
Precision and sensitivity at all sample rates
The Attune® NxT Acoustic Focusing Cytometer enables higher sensitivity when you need it most. You will be able to maintain precise alignment, even at high sample rates of up to 1,000 μL/min. The precise alignment provided by acoustic focusing enables researchers to obtain tighter CVs to better distinguish between dim signals and background resulting in less variation and better signal separation (Figure 3).
|Figure 3. 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 Acoustic Focusing 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 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 cytometer results showed equal or better results even at the highest flow rates.|
Minimal data variation
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 Acoustic Focusing Cytometer, minimal variation in results is seen regardless of sample throughput rate. You no longer need to sacrifice throughput for sensitivity.
- Minimal variation, even at high sample rates
- Less variability in results
- No sacrifice of sensitivity for speed
|Figure 4. Minimal data variation at high sample rates with the Attune® NxT Acoustic Focusing Cytometer. 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 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 width of the G0/G1 and G2/M peaks and CV% remains consistent for the Attune® NxT cytometer, even at the highest sample rate of 1,000 μL/min.|
Rapid detection of rare events
Analysis of rare cell populations requires the collection of high numbers of events in order to attain a reliable measure of accuracy, leading to long acquisition times. The Attune® NxT Acoustic Focusing Cytometer achieves sample throughput at rates over 10 times faster than other cytometers—up to 1,000 μL /min and 20 x 106 events per run, enabling rapid detection of rare events with reliable accuracy while aborting no data.
|Figure 5. 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 interferons 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.|
Dilute your samples, not your data quality
Washing and lysis of red blood cells (RBCs) can cause significant cell loss and damage. Significantly higher sample collection rates allow the Attune® NxT cytometer to deliver a no-wash, no-lyse protocol to minimize cell loss and simplify sample preparation (Figure 6). This feature is particularly useful for samples that are inherently low in concentration. 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 acquired quickly without compromising data. Difficult-to-collect samples like mouse blood and bone marrow, thin-needle aspirates, or any sample with low cell yield can be stained and then diluted without washing or performing RBC lysis. The high collection rate makes acquisition possible—you can run up to 4 mL in just 4 minutes. Because sample preparation steps are eliminated, full-panel testing is possible for precious samples.
Figure 6. Eliminating sample preparation without compromising data quality. Whole blood (5 µL) was stained with antibodies, incubated, and diluted in 4 mL of buffer and analyzed on the Attune® NxT cytometer. 405 nm light is readily absorbed by red blood cells and enables use of the differential side-scatter gating shown in the lower panel, in which a 405 nm vs. 488 nm side-scatter dual parameter plot is used to differentiate the leukocyte and red blood cell populations. From this gated population, a daughter plot of forward and side scatter can be used to identify lymphocyte, monocyte, and granulocyte populations without the need for a fluorescent label.
Software that performs to your specifications
Attune® NxT Software is designed to provide powerful acquisition and analysis using an intuitive interface that is recognizable to all and brings most features that are needed at your fingertips when you need it without needing to scroll through multiple screens. Experiments can be easily set up with settings that can be completely customized and saved for future experiments. Compensation is automated and can be set up using a compensation guide.
The software is designed to maximize the efficiency in performing data analysis with fast refresh rates for large data sets up to 20 million events/sample and allows you to immediately visualize changes on your data plots in real time as you make the adjustment. The software has unique tools to simplify setting up experiments and analyzing data. Reagent selection is simple using the filter configuration manager that provides guidance to help the user match the right reagent to the optimized channel on your instrument by selecting reagents from a drop down of prepopulated or customized reagents, which is then applied to plot labels.
Figures 7 and 8. Examples of the Attune® NxT software interface.