Features common to all Attune flow cytometers

The Attune NxT and Attune CytPix flow cytometers share many of the same features. The Attune CytPix flow cytometer has all the same hardware and functionality as the Attune NxT, with the addition of a high-speed brightfield camera and automated image analysis software. Described below are the features common to both instruments, such as acoustic focusing for sensitivity and fast acquisition, optical design for flexibility and reliability, and clog resistant fluidics. Jump ahead to learn about the imaging and image analysis capabilities of the Attune CytPix. 


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Acoustic focusing for sensitive analysis

Attune Flow Cytometers combine ultrasonic waves like those used in medical imaging with hydrodynamic forces to precisely position cells into a single, focused line in the central axis. Enabling cells to be tightly focused at the point of laser interrogation allows the system to collect more photons, helping to ensure data quality regardless of the sample-to-sheath ratio.

Acoustic focusing

Acoustic focusing (left) precisely positions cells in tight alignment even at higher sample rates resulting in less signal variation and better data quality. Traditional hydrodynamic focusing (right) widens the sample stream core at high sample rates that results in signal variation and compromised data quality.

“When all is said and done, it outperforms [the rest] in that it can analyze 35,000 cells per second with acoustic focusing technology. Its accuracy is not diminished even if you conduct analysis at a high flow rate of 100–1,000 µL/min, which drastically shortens the time required for an experiment.”

Dr. Takashi Satoh,

IFReC Assistant Professor

Osaka University, Japan

Acoustic focusing is useful for cellular imaging on the Attune CytPix instrument, helping to position the cells precisely so that a sharp image is obtained. As demonstrated in these videos, cells imaged without acoustic focusing were fuzzy and out of position, while cells imaged with acoustic focusing were clear and centered in the field of view.

Acoustic focusing positions cells for optimal imaging

Without acoustic focusing (left), beads appear off-center and often blurry. Acoustic focusing (right) reduces lateral position variation, temporal variations, and depth of field limitations to obtain a sharp image. 

Fast sample acquisition speed

Acoustic focusing also lets your lab rapidly acquire high-quality data. Users can achieve sample throughput rates of 12.5 µL/min or 1,000 µL/min, up to 10 times faster than traditional hydrodynamic focusing systems and acquisition speeds of 35,000 events per second. This means processing all samples—including low-concentration and precious samples—more quickly and accurately with minimal loss in quality.

Time to acquire 1 million events

Rapid data acquisition


Compares the time required to acquire 1,000,000 events over three competitor instruments running at maximum sample rates.

Acoustic focusing minimizes variation regardless of the sample rate, so you don't have to make the tradeoff between throughput and sensitivity. This is demonstrated in the cell cycle analysis example below, where it's critical to precisely detect differences in fluorescence intensity between multiple cell populations.

Consistent data collection independent of sample flow rates between 12.5 and 1000 microliters per minute

Minimal data variation


Consistent results were achieved at high sample rates using Jurkat cells fixed and stained with propidium iodide, treated with RNase and analyzed at a concentration of 1 x 106 cells/mL. 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.

“Traditional antibacterial sensitivity testing can take 1–3 days depending on how difficult the bacteria are to grow, isolate, and then test for sensitivity. The Attune NxT allows us, within 1 hour, to tell whether or not the drug will effectively treat the infection. Within 3 hours, accurately assay the minimum drug concentration needed to inhibit bacterial growth.”

Kieran Mulroney,

Biomedical Sciences Researcher

Harry Perkins Medical Research Institute

University of Western Australia

Clog resistant fluidics

To prevent clogging and allow for volumetric analysis, the Attune Flow Cytometers use a positive displacement syringe pump to control sample volume. The system can perform volumetric cell counts in a known volume (gated or total events) and can easily gate out dead cells to count live cells only in a live/dead analysis. Unlike traditional cytometers that operate at a maximum pressure of 15 PSI, Attune Flow Cytometers control pressure at 75 PSI, reducing the likelihood of cell-cell interactions and resulting in clog resistance.


“One of the problems that everyone is familiar with who works in flow cytometry is clogging. Clogging is a thing of the past with this instrument. You have so many samples that you have to run, you can’t wait between samples to clear everything out.”

Bruno Sainz,

CLIP Investigator

Universidad Autónoma de Madrid

Madrid, Spain

Volumetric sample entry


Using positive displacement, the syringe pump works like a pipette. Tension is created with the plunger pushing down (A). The tension is released as the plunger rises with the sample (B). Clogging is less likely to occur under this high and controlled pressure environment.

“We needed to have a flow cytometer that would allow us to take samples from tumors that had tended to clog other machines. Therefore the Attune NxT is the flow cytometer of choice for our application. The Attune NxT has a number of characteristics that are critical for our research… program. Not only does it have acoustic focusing, which will allow us to go at higher sample throughput, but it also has a larger flow cell, so when we’re looking at tumor cells and isolating cells from those tumors we don’t have to worry about clogging and having to spend a lot of downtime getting the instrument up and running again.”

Charles Prussak,

PharmD, PhD

Director of the Cell Therapy Translational Laboratory (CTTL)

University of California San Diego

Flexible optical design

Attune Flow Cytometers are configurable with up to 4 spatially separated lasers and 16 parameters. Spatial separation provides flexibility for multicolor panel design and streamlines compensation.  The system offers superior speed with acquisition rates of up to 35,000 events per/second with high sensitivity to meet a range of research requirements.


High sensitivity distinguishes between dim signals and background, resulting in less variation and better signal separation. Fluorescent resolution coefficient variation is less than 3% for a single peak, and predicted MESF is ≤80 (FITC), ≤30 (PE), ≤70 (APC). Sensitivity in comparison to competitive systems is described below.

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 were equal to or better than those from the conventional flow cytometer, even at the highest flow rate.

The compact size of the Attune Flow Cytometer also provides the flexibility of using it within a biosafety hood. This helps avoid contamination or infection when working with hazardous or unknown samples.

Lasers designed for stability

Attune Flow Cytometers feature a novel optical design that delivers first-class reliability and superior performance over time. The flat-top beam profile of the solid-state lasers minimizes the effects of changes in fluidics or optics, which in turn can lead to instability or alignment issues and instrument downtime.


Laser misalignment is a major concern with users of conventional flow cytometers. The flat-top lasers used in the Attune Flow Cytometers have an intensity profile that allows a wider window of alignment over Gaussian lasers used in traditional systems.  The flat-top lasers also have a higher tolerance for misalignment that allows them to maintain high sensitivity and low CVs.

Emission profile of lasers


Gaussian laser used in traditional cytometers (left) with misalignment and flat-top laser used in the Attune Flow Cytometers (right) showing proper alignment if shifted.

Rare event detection

Detection of rare events requires acquisition of high numbers of cells to attain a reliable measure of accuracy. Attune Flow Cytometers allow dilute samples to be processed quickly at sample input speeds of up to 1 mL/min, significantly faster than conventional cytometers that support maximum sample input rates of 60–100 µL/min. Acoustic focusing thus offers a unique combination of speed and quality, cutting the time to collect rare events significantly over long acquisition times.



Collecting more than 1 million live cells and detecting a rare population of dendritic cells


Plasmacytoid dendritic cells (pDCs) are identified using 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.

Attune Flow Cytometers support both high speed of acquisition and high sensitivity to enable easy detection and phenotypic and functional characterization of rare cells. This step-by-step strategy for detecting rare events in our Flow Cytometry Learning Center will help you think through the best approach for pre-analytical, analytical, and data analysis phases of your research.

Attune CytPix Flow Cytometer features

The distinguishing features of the Attune CytPix Flow Cytometer are a high-speed brightfield camera and automated image analysis for morphometric data.

High-speed camera

The camera captures images of events at a rate up to 6,000 images/second and enables users to visually verify events recorded by the detector. Users can view event images in real-time to confirm sample quality, optimize protocols, and record the morphology of each cell population identified for analysis. This is crucial in cell therapy applications but can be applied to any flow cytometry sample.


Imaging can be added without changing existing flow cytometry protocols or reducing throughput. The Attune CytPix Flow Cytometer can capture clear images, even while maintaining standard flow cytometry acquisition speeds. Imaging also benefits from acoustic focusing, which helps to position the cells so that a sharp, centered image is obtained.

Consistent image quality even at high flow rates


Acoustic focusing and a high-speed camera combine to image these CAR T cells consistently at low or high flow rates. Focus and camera settings are easily adjusted to meet experimental requirements.

By using the image gallery view, users can scan cell populations rapidly for outliers. In this view, doublets and other aggregates stand out, even in cursory review. With the Attune CytPix Flow Cytometer, users can highlight structural features of large populations in record time. This allows adjustment of gates to include cells of interest while excluding aggregates, unwanted cells, and debris.

High-level image gallery view

Early log phase Jurkat cells were acquired unfiltered on the Attune CytPix Flow Cytometer at 200 µL/minute, >105 cells/mL. Image gallery view was used to rapidly scan cellular events.

Morphological information from images adds to the richness of flow cytometry data over multiplexed staining alone. For example, the figure shows an otherwise conventional apoptosis assay using Annexin V and propidium iodide (PI), with added cell imaging to characterize cells in each population, revealing morphologically distinct features. These insights could not have been gained from flow cytometry data alone.

Morphological characteristics of apoptotic cells.

Jurkat cells were incubated with 10 µM camptothecin for 4 hours at 37ºC to induce apoptosis. Samples were stained with Annexin V and PI and acquired on the Attune CytPix Flow Cytometer at 100 µL/minute. From the singlet population, gating strategies identified three cell subpopulations. About 50% of apoptotic live cells (Annexin V⁺PI-, bottom right) showed some form of apoptotic body such as blebs. About 25% of apoptotic dead cells (Annexin V⁺PI⁺, top right) showed increased cell surface granularity, and there were more partial cells. About 10% of healthy cells (Annexin V-, bottom left) showed apoptotic bodies (though not as severe as those observed among Annexin V⁺ cells). These healthy cells were also morphologically diverse and included some doublets despite upstream singlet gating. Morphological features in the images are indicated by black arrows.

Automated image analysis for morphology

The Attune Cytometric Software on the Attune CytPix Flow Cytometer analyzes and interprets cell features from images into morphometric parameters using models pre-trained on leukocytes and beads.  Combined with captured images and flow data, these parameters enable researchers to gain novel insights into their samples. Gates can be adjusted to exclude aggregates, coincident events, debris and unwanted cells.


The software automates image analysis at a rate of up to 1,000 images/second and can be managed by users in a processing queue that runs in the background of the software. These extended image-derived parameters provide data to confirm singlets with cell count (Particle Count) and morphology features such as roundness (Circularity), size (Area Square), shape (Eccentricity) and complexity (Entropy).  Gating on these extended parameters allows you to quickly and accurately identify populations of interest to confirm gating strategy with little or no manual review. You can use back-gating to scan the panel of full resolution images and correlate what you see with scatter, fluorescence or image-based parameters to any population on the dot plot. Enhance the quality of your data and feel confident when gating with powerful data-driven cell analysis.