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.
For the researcher who prefers to avoid color compensation, the 14 colors spread across the 4 independent laser lines allow popular dye combinations to be used with minimal or no compensation.
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 choose dyes and detection channels that are well separated spectrally and do not have significant overlap, requiring minimal compensation (Figure 1).
Figure 1. 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.
With the option to be configured with up to 4 lasers and 14 colors for multiparameter analysis (Figure 2), the Attune NxT flow 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 (see Figure 3).
Figure 2. 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.
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: mCherry, the popular monomeric red-fluorescent protein with superior brightness and photostability; mKate, known for its fast maturation rate as well as its high pH stability and photostability; and mOrange2, a bright monomeric orange-fluorescent protein. 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 3 shows detection of TagBFP, emGFP, YFP, mOrange2, TagRFP, mKate, and mCherry using the Attune NxT flow cytometer.
Figure 3. 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.
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. 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 Flow Cytometer allows dilute samples to be processed quickly at sample input speeds of up to 1 mL/min. Conventional cytometers utilize traditional hydrodynamic focusing technology that allows for maximum sample input rates of 60–100 µL/min, limiting detection of rare cell populations due to the severely long acquisition times required to collect enough cells for analysis. By combining acoustic and hydrodynamic focusing, the Attune NxT cytometer can analyze cells at sample input rates of 500 µL/min or 1 mL/min, allowing for quick collection time in experiments involving detection of rare events (Figure 4).
Figure 4. 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.
Save time—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 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 5).
Figure 5. 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 6). You no longer need to sacrifice throughput for sensitivity.
Figure 6. 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.
The Attune NxT flow 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 7).
Figure 7. 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.
Standard methods for isolating and detecting leukocytes in whole blood are time consuming and involve significant sample manipulation prior to analysis. These steps can lead to sample loss and alterations in cell physiology. Significantly higher sample collection rates allow the Attune NxT flow cytometer to deliver a no-wash, no-lyse protocol to minimize cell loss and simplify sample preparation in whole blood immunophenotyping (Figure 8). Higher sample collection rates are also beneficial for samples that are inherently low in concentration (i.e., cerebrospinal fluid (CSF) and stem cells) and for precious samples (i.e., mouse blood, bone marrow, and thin-needle aspirates) that require that entire sample be analyzed to collect sufficient events.
Figure 8. Identification of leukocytes in human whole blood using violet side scatter on the Attune NxT flow cytometer.(A) Using conventional blue 488 nm forward scatter (FSC) and side scatter (SSC) does not allow resolution of leukocytes in whole blood. Backgate analysis using fluorescently labeled antibodies specific for leukocytes (pink) and red blood cells (blue) demonstrates this problem. (B) Resolution of leukocytes from red blood cells in whole blood is improved by incorporating violet 405 nm SSC using the Attune NxT No-Wash No-Lyse Filter Kit. (C) Backgate analysis using antibodies against the red blood cell marker glycophorin A and antibodies against the leukocyte marker CD45 demonstrates the ease of identifying leukocytes in human whole blood, in contrast to the FSC and SSC analyses in (A) and (B). (D) Using both violet and blue SSC allows identification of leukocytes in whole blood, which is corroborated by the backgate analysis using glycophorin A and CD45 labeling depicted in (C), and demonstrates the different scatter properties of leukocytes and red blood cells when using violet SSC. (E) When leukocytes are gated based on violet light scatter properties, the three main leukocyte cell populations in human blood can be distinguished: lymphocytes, monocytes, and granulocytes.