The Attune® Acoustic Focusing Flow Cytometer—How it Works

The Attune® Acoustic Focusing Flow Cytometer is designed to deliver higher sensitivity when you need it most. You’ll be able to maintain precise alignment, even at high sample rates (up to 1 mL/min). Alternatively, you can use a slower rate to collect more photons when analyzing dim signals—this translates to better separation and better clarity of your results. Data obtained with the Attune® Acoustic Focusing Cytometer are more precise (narrower fluorescence peaks) than data obtained with traditional hydrodynamic focusing instruments (Figure 1), resulting in greater separation and easier data interpretation. Switching to highest sensitivity mode, the Attune® Flow Cytometer was able to detect beads that were too faint for the conventional flow cytometer’s detectors (Figure 2), demonstrating sensitivity equal to or better than a conventional flow cytometer.

• Download the poster: Attune® cytometer sensitivity and throughput
  
• Download the tech note: Comparison using a hydrodynamic flow cytometer and 8-peak calibration particles
• Download the tech note: White blood cell scatter resolution and immunophenotyping using whole blood

click to enlarge		Figure 1. Greater peak separation with phosphospecific antibody staining. Intracellular target detection can be challenging. Jurkat T cells were stimulated with anti-CD3 and anti-CD28 antibodies, then fixed and permeabilized before staining with a PE-labeled phosphospecific antibody. The sample was run on the Attune® Flow Cytometer and a conventional hydrodynamic cytometer and the data were compared. The plots show an overlay of the data from stimulated and unstimulated samples from each system.
click to enlarge		Figure 2. Sensitivity through control of cell transit times. Fluorescent microspheres (Spherotech Rainbow 3.2 μm) were run on a high-end conventional flow cytometer and on the Attune® Acoustic Focusing Cytometer using a 488 nm blue laser (top row) and a 405 nm violet laser excitation (bottom row). The conventional flow cytometer was run at its highest sensitivity setting, at a sample input rate of 15μL/min (left column). The Attune® Flow Cytometer was run at 2 different settings: at its standard sensitivity setting, at a 100μL/min sample input rate (middle column), and at its highest sensitivity setting: with a 100 μL/min sample input rate and 4x increase in the amount of time the particles are illuminated by the laser (right column).

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The Attune® Acoustic Focusing Cytometer achieves sample throughput at rates over 10 times faster than other cytometers—up to 1,000 μL per minute. The Attune® Flow Cytometer is designed to collect up to 20 million events per run and has adjustable collection rates of 25–1,000 μL/min. Table 1 below compares the acquisition times with a hydrodynamic focusing cytometer or the Attune® cytometer using a blood sample from an individual with aplastic anemia, each with a stop gate set on 1 million granulocyte events.

Table 1.  Comparison of Acquisition Times.

Figure 3. Rare event detection of 0.2% dendritic cells from mouse splenocytes. Plasmacytoid dendritic cells (pDCs) are a specialized cell type 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 7.5x107 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.

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With volumetric analysis, the Attune® Acoustic Focusing Cytometer enables absolute counting and eliminates the need to use expensive counting beads. Figure 4 shows the identification of CD34+ cells from peripheral blood with an acquisition time of 4 minutes 28 seconds.

• Download Absolute Cell Counting app note

• Download the Attune® cytometer Rare Event Detection for Clinical Research poster

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Figure 4. Identification of CD34+ cells from peripheral blood. Peripheral blood from a normal donor was stained and run on the Attune® Flow Cytometer at a collection rate of 1,000 μL/min with a stop gate set at 500,000 total cells. A rare population of 0.045% CD34+ cells (red box) was identified from within the population of cells.

Significantly higher sample collection rates allow the Attune® Flow Cytometer to untilize a no-wash, no-lyse protocol that avoids cell loss and results in additional time savings by reducing the number of sample preparation steps.

Dilute samples like cerebrospinal fluid (CSF), stem cells, or any sample with low cell numbers can take a long time to acquire. The Attune® Flow Cytometer allows researchers to intentionally dilute samples in place of a wash/centrifugation step. In the case of very low amounts of available sample, dilution to 1 mL or more can help preserve the sample while not significantly affecting data acquisition time.

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. High-rate collection makes acquisition possible—you can run up to 4 mL typically in just four minutes. You don’t lose any sample through wash steps, and full panel testing is possible for all precious samples. All sample preparation steps can be eliminated without compromising the data by diluting the sample (using a rate of 500 μL/min) (Figure 5).

Download application notes:

• A No-Lyse, No-Wash Method for Mouse Immunophenotyping
• A No-Lyse, No-Wash Method for Immunophenotyping Human Blood Cells
• Compare a No-Lyse, No-Wash method Using Red or Violet Laser

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Figure 5. Eliminating sample preparation without compromising data. Normal whole blood (100 μL) was labeled with CD45 Pacific Blue, CD3 Alexa Fluor® 488, and CD4 PerCP-Cy®5.5 direct conjugates. After 15 minutes of incubation, 5 μL of the stained whole blood was diluted into 4 mL PBS, and data were acquired on the Attune® Flow Cytometer without RBC lysis or washing. A fluorescence threshold was set on the Pacific Blue™ dye to include only CD45-positive cells. A gate was created around the lymphocyte population in a CD45 vs. SSC plot (A) to analyze the mutually exclusive CD4 and CD8 populations (B).

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