Attune® Cytometer front view - Enlarge
The Attune® Acoustic Focusing Flow Cytometer gives you both high precision and sensitivity at any rate of throughput. You can control your sample concentration, the flow rate, the number of photons you detect, the length of your experiment, the number of samples you run, and more. All this translates to dependable results—faster and easier than ever before.
The Attune® Acoustic Focusing Flow Cytometer delivers 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 Attune® Sensitivity and Throughput Poster
|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 compared. The plots show an overlay of the data from stimulated and unstimulated samples from each system.|
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 100 µL/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).
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® Acoustic Focusing 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.
|Instrument (collection rate)||Time to acquire 1 million granulocyte events||Relative rate compared to hydrodynamic focusing|
|Hydrodynamic focusing cytometer (high)||63 min 33 sec||--|
|Attune® Acoustic Focusing Cytometer (200 µL/min)||13 min 20 sec||4.8x faster|
|Attune® Acoustic Focusing Cytometer (500 µL/min)||5 min 47 sec||11.0x faster|
|Attune® Acoustic Focusing Cytometer (1,000 µL/min)||3 min 13 sec||19.7x faster|
In addition, the Attune® Flow Cytometer enables rapid detection of rare events without aborting data, resulting in better data quality and reliable accuracy. The Attune® Cytometer was used to analyze more than 1 million live cells, resulting in the detection of a rare population (0.2%) of dendritic cells from within a population of mouse splenocytes (FIgure 3).
|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 7.5x10^7 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 Cytometer - Absolute Cell Counting Without Beads
With volumetric analysis, the Attune® Acoustic Focusing Cytometer enables absolute counting and eliminates the need to use expensive counting beads. Figure 4 below shows the identification of CD34+ cells from peripheral blood with an acquisition time of 4 minutes 28 seconds.
|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 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
|Figure 5. Eliminating sample preparation without compromising data. Normal whole blood (100 µL) was labeled with CD45 PacificBlue™, 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).|
The Attune® Cytometric Software can be downloaded without licensing fees. The software can be added to any desktop or laptop computer at your institution without any additional costs. Results can easily be analyzed at your convenience at your own computer, allowing the next user to run experiments on the Attune® cytometer.
The Attune® Cytometric Software is designed to provide powerful user-defined analysis using an intuitive interface for simplified experimental analysis. Templates can be built around specific applications and saved for consistent experimental design. Compensation is automated or user-defined and can be set up using a compensation guide. Utilities such as quick-save, drag-and-drop, and copy-and-paste provide rapid manipulation with commonly used functions. Experiments can be easily set up with automated settings that can be completely customized and saved for future experiments.