flow cytometry reinvented

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1 Flow cytometry flow cytometry reinvented The Attune Acoustic Focusing Cytometer Precision and sensitivity at all speeds for rare events and precious samples

2 The Attune Acoustic Focusing Cytometer Precise high collection rates without compromising CVs Fast rare-event detection with dramatically shorter acquisition times Simple no-lyse, no-wash method eliminates cell loss With the Attune Acoustic Focusing Cytometer, you get both high sample input rate and high precision when analyzing precious samples or detecting rare events. You control your sample concentration, flow rate, the number of photons you detect, the length of your experiment, and more. You get the most out of your cells with dependable results. But best of all, the Attune cytometer makes it easy. Precision and sensitivity at all sample throughput rates The Attune Acoustic Focusing Cytometer enables higher sensitivity when you need it most. You ll be able to maintain precise alignment, even at high sample rates of up to 1 ml/min. And because the Attune cytometer is not limited by physical flow constraints, you can control the amount of time cells spend in the lasers path. This means you can use a slower rate to collect more photons when analyzing dim signals, translating to better clarity of your results and better separation (Figures 1 and 2). Hydrodynamic focusing instrument % Attune cytometer, normal sensitivity mode % of Max Note: increased precision as seen by the tighter populations % PE-A BL2-A:BL2-A 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 cytometer and a hydrodynamic focusing cytometer and the data compared. The plots show an overlay of the data from stimulated and unstimulated samples from each system. The data obtained with the Attune Acoustic Focusing Cytometer have higher precision (narrower peaks), resulting in greater separation and easier data interpretation.

3 Conventional flow cytometer (Highest sensitivity setting) Attune cytometer (Standard sensitivity setting) Attune cytometer (High sensitivity setting) 675 LP 675 LP 675 LP PE-Cy 5 585/ PE-Cy T 5 585/ / PE-Cy Pacific Orange TM Pacific Orange TM Pacific Orange TM 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 15 μl/min (left column). The Attune 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 cytometer standard settings resulted in sensitivity equal to or better than the conventional flow cytometer, while the high sensitivity of the Attune instrument resulted in detection of the faintest beads not detected by the conventional flow cytometer. Rare events detection 10 times faster The Attune Acoustic Focusing Cytometer achieves sample throughput at rates over 10 times faster than other cytometers up to 1,000 µl per minute, enabling rapid detection of rare events with reliable accuracy while aborting no data (Table 1, Figure 3). The Attune cytometer is designed to collect up to 20 million events per run and has adjustable collection rates of 25 1,000 µl/min. CD45R/B220-PE fluorescence Gated on live CD19 - cells at collection rate of 500 ul/min CD317-Alexa Fluor 488 fluorescence Table 1. Comparison of acquisition times with a hydrodynamic focusing cytometer or the Attune Acoustic Focusing Cytometer, using a blood sample from an aplastic anemia individual, each with a stop gate set on 1 million granulocyte events. Instrument (collection rate) Hydrodynamic focusing cytometer (high) Attune Acoustic Focusing Cytometer (200 µl/min) Attune Acoustic Focusing Cytometer (500 µl/min) Attune Acoustic Focusing Cytometer (1,000 µl/min) Time to acquire 1 million granulocyte events Relative rate compared to hydrodynamic focusing 63 min 33 sec 13 min 20 sec 4.8x faster 5 min 47 sec 11.0x faster 3 min 13 sec 19.7x faster Figure 3. 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, CD Four-color staining of mouse splenocytes included CD19 Pacific Blue, CD317 Alexa Fluor 488, CD45R/B220-PE direct conjugates, and SYTOX AADvanced TM Dead Cell Stain. A gate was made on live cells using SYTOX AADvanced TM 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.

4 SSC Attune sample rate: 1,000 µl/min CD CD34 Figure 4. Identification of CD34 + cells from peripheral blood. Peripheral blood from a normal donor was stained and run on the Attune Acoustic Focusing 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 within the population of cells with an acquisition time of 4 minutes, 28 seconds. In addition, the Attune cytometer provides direct cell counts without the need for expensive counting beads with volumetric analysis (Figure 4, Table 2). Table 2. Comparison of absolute cell counting with a hydrodynamic focusing cytometer or the Attune Acoustic Focusing Cytometer. Instrument Sample Flow Rate (µl/ min) Acquisition Time Percentage of CD34 + Cells CD34 + Cells/µL, Direct Measurement Attune Acoustic Focusing Cytometer 1,000 4 min, 28 sec 0.05% 0.05 High-end conventional cytometer min, 46 sec 0.02% N/A Dilute your samples, not your data quality Washing and lysis of red blood cells (RBCs) can cause significant cell loss and damage [1]. Significantly higher sample collection rates allow the Attune cytometer to deliver a no-wash, no-lyse protocol to minimize cell loss and simplify sample preparation (Figure 5). 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 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. High rate collection makes acquisition possible you can run up to 4 ml in just four minutes. No sample loss occurs from sample preparation, and full panel testing is possible for all precious samples. 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 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). By diluting the sample and using a rate of 500 µl/min, all sample preparation steps can be eliminated without compromising the data.

5 Cell cycle analysis using the Attune Acoustic Focusing Cytometer 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 Acoustic Focusing Cytometer, minimal variation in results is seen regardless of sample throughput rate. You no longer need to sacrifice throughput for sensitivity (Figure 6). Minimal variation, even at high sample rates (Figures 6 and 7) Less variability in results (see changes in %S phase from Table 1) No sacrifice of sensitivity for speed %CV C-Low (12 µl/min) C-Med (35 µl/min) C-High (60 µl/min) Attune (25 µl/min) Attune (100 µl/min) 60 µl/min G O /G 1 CV 1,000 µl/min Attune (200 µl/min) Attune (500 µl/min) Attune (1,000 µl/min) Figure 7. Percent CV of G 0 / G 1 at different flow rates on the Attune cytometer and a competitor (C) at different sample rates. Note the minimal change in variability (%CV) for the Attune Acoustic Focusing Cytometer, even at a high sample rate. A High-End Hydrodynamic Focusing Cytometer Low - 12 µl/min B Attune Acoustic Focusing Cytometer 25 µl/min Five other cell types were compared to a high-end hydrodynamic focusing cytometer, including ModFit analysis in each case. The sample throughput rate of 25uL/min was used for the analysis (Figure 8). C Medium - 35 µl/min D 200 µl/min Ramos B Cells A Hydrodynamic System G0G1 CV = 3.35% B Attune Acoustic G0G1 CV = 3.11% E High - 60 µl/min F 1,000 µl/min KG-1 a Myeloblast Cells C Hydrodynamic System G0G1 CV = 5.53% D Attune Acoustic G0G1 CV = 3.58% HL60 Promyeloblast Cells E Hydrodynamic System G0G1 CV = 6.10% F Attune Acoustic G0G1 CV = 2.14% Figure 6. Minimal data variation at high sample rates with the Attune Acoustic Focusing Cytometer. Jurkat cells were fixed and stained with propidium iodide, treated with RNase, and analyzed at a concentration of 1 x 10 6 cells/ml on a high-end instrument that uses hydrodynamic focusing, and on the Attune Acoustic Focusing Cytometer at different sample rates. The left peak in all graphs reflects cells in G 0 /G 1 phase, while the right peak reflects cells in G 2 /M phase. Note that as sample rates increase on the instrument that uses hydrodynamic focusing, the width of the G 0 / G 1 and G 2 /M peaks increase, whereas for the Attune cytometer the peaks are relatively stable, even at the highest sample rate of 1,000 µl/min. U266 Myeloma Cells G Hydrodynamic System H Attune Acoustic G0G1 CV = 7.17% G0G1 CV = 4.41% ST486 B Cells I Hydrodynamic System J Attune Acoustic G0G1 CV = 5.84% G0G1 CV = 2.81% Figure 8. Attune Acoustic Focusing Cytometer exhibits greater precision. Data collected on the Attune cytometer exhibits lower coefficients of variation (%CV) and more distinct cell populations when compared to a high-end hydrodynamic focusing flow cytometer.

6 What is acoustic focusing cytometry? The Attune Acoustic Focusing Cytometer is the first cytometer that uses ultrasonic waves (over 2 MHz, similar to those used in medical imaging), rather than hydrodynamic forces, to position cells into a single focused line along the central axis of a capillary (Figure 9). Acoustic focusing is largely independent of the sample input rate, enabling cells to be tightly focused at the point of laser interrogation regardless of the sample-to- ratio (Figure 10). This, in turn, allows slowed cell velocity to collect more photons for high-precision analysis at unprecedented volumetric sample throughput. The Attune cytometer accomplishes all this without high velocity or high volumetric fluid, which can damage cells. In addition, volumetric syringe pumps enable absolute cell counting without beads minimizing cost and sample preparation time. In contrast, cytometers that use hydrodynamic focusing maintain the same sample speed at all flow rates, causing cells to lose focus as the sample core widens to increase flow rate. A. Acoustic focusing = better precision Acoustically focused sample stream Laser (cross-section) Acoustic focusing = OFF Sample is unfocused Acoustic focusing = ON Sample is focused Figure 9. Acoustic focusing in action. Fluorescent microspheres were applied to the capillary system of an acoustic focusing cytometer. Beads flow through randomly without any acoustic focusing (left). With the application of acoustic focusing, the beads are focused into a single line (right). No more tradeoffs Hydrodynamic focusing cytometers have many limitations. As a result, users often have to sacrifice: Throughput for sensitivity Features for ease of use Performance for price Power for footprint B. Traditional hydrodynamic focusing: compromised of data quality Hydrodynamic core device Laser (cross-section) Acoustic focusing technology keeps cells within a confined focal point, so these tradeoffs are not required. Figure 10. Acoustic focusing vs. traditional hydrodynamic focusing. (A) In acoustic focusing, cells remain in tight alignment even at higher sample rates. With this tight alignment, cells pass through the laser beam at its optimal focal point, resulting in less signal variation and improved data quality. (B) In traditional hydrodynamic focusing, increasing the sample rate results in widening of the sample core stream. The speed at which cells pass through the laser is not changed, and is determined by the speed of the fluid flow. Cells are distributed throughout the sample core stream because of reduced differential pressure between sample stream and stream, resulting in reduced cell focusing. Cells are not in tight alignment as they pass through the laser beam, resulting in increased signal variation and compromised data quality.

7 Ideal for both standard and specialized research applications Most standard applications for flow cytometry have been tested on the Attune cytometer, including: Live/dead cell discrimination Cell cycle analysis Cell proliferation assays Basic phenotyping (up to 6 colors) Rare-event detection Apoptosis Phagocytosis Detection of phosphoproteinsphagocytosis Other intracellular markers Many standard cell types have been tested, including: Jurkat cells HL60 promyoblast cells U266 myeloma cells, Mouse splenocytes Mouse blood HeLa human cervical carcinoma cells Bovine pulmonary artery epithelial (BPAE) cells 3T3 mouse embryo fibroblast cells Chinese hamster ovary (CHO) cells Human mesenchymal stem cells (hmsc) E. coli, S. aureus Human platelets Human whole blood Data have also been collected in specialized research areas such as oceanography and microbiology. Cells up to 100 μm in diameter have been run without clogging our 200 μm flow cell, with a minimum particle size specification of 1 μm; however, researchers are constantly pushing these limits with the Attune cytometer. Service and support The Attune Acoustic Focusing Cytometer is backed by Life Technologies worldwide technical support and service programs. We are focused on delivering personalized service from the time our sales representative walks through the door and throughout the life of your Attune instrument. The Attune cytometer is fully supported for one year with our extensive service plan, which includes: Comprehensive training Application and assay support Worldwide technical service Preventive maintenance Extended service plans are also available. For more information about available service plans, go to 1. Gratama JW, Menéndez P, Kraan J, Orfao A (2000) Loss of CD34(+) hematopoietic progenitor cells due to washing can be reduced by the use of fixative-free erythrocyte lysing reagents. J Immunol Methods 239:13 23.

8 Attune Acoustic Focusing Cytometer Software that performs to your specifications User-defined analysis, flexible setup Attune Cytometric Software is designed to provide powerful user-defined analysis using an intuitive interface for simplified experimental analysis (Figure 11). 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. No software licensing fees 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. Instrument controls Instrument status Control sample flow rate Select sensitivity level Run startup/shutdown protocol System performance tracking by date Visualization tools Plot overlay or side-by-side graph comparison Standard or custom gate shapes Histograms, dot plots, density plots Customize statistics Copy and paste plots into reports (Microsoft Word or PowerPoint software) Analysis setup Save protocol templates Automated or user-defined compensation Raw data capture Data display in linear, log, or lin/lg scales Set universal compensation across multiple data sets File format compatibility Save data and images in PDF format for reports Data format in standard FCS 3.0 for compatibility with third-party analysis tools (i.e., FlowJo, ModFit LT, FCS Express software programs) Statistics in.csv format

9 Instrument performance tracking The Attune Cytometric Software provides automated baseline calculation and performance test functions with minimal user interactions. Performance tracking is facilitated through the reports function in the software. Instrument performance tracking is critical for collecting and analyzing accurate experimental data. The Attune Performance Tracking Beads are designed for use with the Attune Cytometric Software to automatically characterize, track, and report performance measurements of the Attune Acoustic Focusing Cytometer (Figure 12). The beads are used to define a baseline and conduct daily measurements of the cytometer. Vials contain enough beads for 50 measurements, and specific information for each lot is downloaded to the Attune Cytometric Software prior to use. Performance tracking for the Attune cytometer includes a comprehensive set of procedures to monitor the daily performance of the instrument. The performance tracking process involves: Running performance tracking beads Monitoring changes in the coefficient of variation and in the associated PMT voltage Tracking linearity of instrument performance Evaluating the detector sensitivity and background over time Automatic setting of laser delay Instrument performance is affected by the lasers, optics, and fluidics of the instrument. For optimal instrument performance, it is critical to follow the manufacturer s recommended schedules for maintenance. Figure 11. Example of Attune Cytometric Software. The instrument control panel on the left side of the screen is used for experiment setup and control of laser selection, optics, and fluidics (flow rates). The main frame of this screen shows data analysis plots, while the right panel shows sub-file organization. Figure 12. Example of Levey-Jennings plot showing Attune cytometer performance over time. This report is generated using data from performance tracking beads.

10 NOTES

11 Ordering information Description Quantity Part number Attune Acoustic Focusing Cytometer (includes computer, monitor, startup solutions, installation, and warranty) Attune Focusing Fluids, 1X solution, 1 L Attune Focusing Fluids, 10X solution, 1 L Attune Focusing Fluids, 1X solution, 6 x 1 L Attune Wash Solution Attune 10X Shutdown Solution Attune Performance Tracking Beads (5 x 10 6 beads/ml) Life Technologies offers a breadth of products DNA RNA protein cell culture instruments For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. LIMITED USE LABEL LICENSE: No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Printed in the USA. CO