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The Attune CytPix Flow Cytometer is an advanced cell analyzer that shares common features with the Attune NxT model.
Its distinguishing feature is a high-speed brightfield camera that records images of individual events as they pass through the flow cell. The camera and Attune Cytometric Software help to ensure that the events you analyze are single cells as opposed to doublets, clumps, or debris. This is crucial in cell and gene therapy research applications, but is useful in almost any flow cytometry experiment to help researchers understand the morphology of each cell population identified for analysis. The images can also aid in identifying debris and optimizing protocols.
As samples are acquired on the Attune CytPix Flow Cytometer, the high-speed camera captures and stores images of a sampling of events. For greater flexibility, Attune Cytometric Software lets you adjust the image capture frequency as needed, and can capture up to 6,000 images per second depending on the flow rate and image size. Acoustic focusing helps to position the cells so that a sharp image is obtained.
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.
Optical focus is maintained regardless of the sample flow rate. You can image events at flow rates up to 1,000 µL/minute, and adjust the focus, imaging window, and illumination settings to your target cell type.
The Attune Cytometric Software on the CytPix Flow Cytometer analyzes and interprets cell features from images into quantitative 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-based 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.
Imaging-enhanced flow cytometer applications include almost any study that can benefit from understanding the morphology of each cell population identified. The imaging capability lets you look more deeply into results to:
Confirm gate accuracy in real time
Use images to adjust gates and camera settings, and exclude atypical cells and debris for more robust gating
Further characterize cell populations
Document morphologically distinct populations in existing protocols such as apoptosis detection
Capture cell-to-cell interactions
Visualize and differentiate coincident events
Visualize structural features of large populations
Use high-throughput, detailed photographic evidence
Discover opportunities for analysis
Based on fluorescence, scatter, and morphological features
Measure derived image parameters
Using the image analysis feature of the Attune Cytometric Software 6.0
High-throughput quality control
Detect quality issues quickly by adding rapid imaging to cell culture QC workflows, and monitor changes in cell morphology as the plate is processed
Optimize gating strategy. Even robust manual singlet gating is error-prone and remains a subjective decision point in almost all flow cytometry assays. Imaging can be used to confirm and adjust gates to include only single cells of interest.
Here, an experienced user has gated singlets confidently. After evaluating the manual singlet gate, the CytPix image-derived parameter ParticleCount reveals this gate contains more than 4% aggregates.
Perhaps most importantly, these events contain cells of clearly different phenotypes which could lead to incorrect conclusions regarding double positive events (especially in rare populations).
Characterize cell populations. Morphological information from images can add to the richness of flow cytometry data. For example, the figure shows an otherwise conventional apoptosis assay using Annexin V and PI, adding cell imaging to characterize cells in each population to reveal morphologically distinct features. These insights could not have been gained from multiplex staining alone.
Analyze cell-to-cell interactions. Imaging can even show interactions between cells. In the figure, engineered CAR-T immunotherapy cells were co-incubated with Ramos (lymphoma) cells and stained, acquired, and imaged on the Attune CytPix Flow Cytometer. Images from quadrant Q2 (positive for both stains, acquired as a single event) show the CAR-T cells visibly targeting the Ramos cells, clear evidence of engineered cell potency.
We previously demonstrated the power of imaging CAR-T/Ramos cell interactions. Let’s look at just the population of greatest interest, the double positive events, to learn more. We can now use extended image-derived parameters (circularity vs skewness of intensity) to further examine the features of these populations and refine gating on these events, increasing data robustness. Here, we show that by gating strategies using image-based quantitative parameters can distinguish interacting cells from coincident events to more accurately analyze interacting cells.
Backgating imaged cells on the Attune CytPix Flow Cytometer also allows you to use morphological features to discover interesting subpopulations that would not be apparent from flow cytometry data alone.
For example, E. coli cells incubated over time develop into two types of colony-forming units (CFUs): short CFUs that resemble single cells, and elongated structures with incomplete fission rings, representing incomplete constriction at each approximate cell length. Neither a traditional singlet gate (SSC-A vs SSC-H) nor a fluorescence gate (SSC vs nucleated stain) sufficiently separates these populations. But with the Attune CytPix imaging-enhanced flow cytometer, you can view and group the images and gate the CFU types based on their morphological characteristics.
Discrimination of two E. coli CFU types. E. coli cells were incubated overnight at 37ºC followed by 3 days at 4ºC. Samples were acquired on the Attune CytPix Flow Cytometer at 100 µL/minute. From the images, two types of CFUs were identified: (A) short colonies resembling single cells and (B) elongated structures with incomplete fission rings. Representative images from each population are shown. Backgating on the selected images demonstrated that the two populations are distinct on FSC vs SSC dot plots (orange dots, left).
Cell culture QC. Adding rapid imaging to quality control (QC) workflows can detect and track down cell culture issues early in the process. In one lab, for example, a routine passage check of a Ramos (lymphoma) cell culture observed reduced cell counts and survival despite appearing confluent. Further investigation revealed substantial microbial contamination, but when and where did it begin?
Because the cell line had previously been analyzed on the Attune CytPix Flow Cytometer, the researchers went back to the images and were able to document the microbial infection at least five days earlier. At that time, the early signs were dismissed as debris, but the retrospective evaluation demonstrated shared characteristics with the problematic cells in culture. Tracing the infection helped the lab establish additional laboratory procedures for screening and protection of assay-critical cell lines.
Lasers | Laser configuration | Number of detection channels for included lasers | Total detection channels* | Catalog number | |||
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Violet, 405nm | Blue, 488nm | Yellow, 561nm | Red, 637nm | ||||
2 | Blue/yellow
|
✝ | 3 | 4 | ✝ | 9 | A51842 |
Blue/red
|
✝ | 4 | ✝ | 3 | 9 | A51840 | |
Blue/violet
|
4 | 4 | ✝ | ✝ | 10 | A51841 | |
Blue/violet 6
|
6 | 3 | ✝ | ✝ | 11 | A51843 | |
3 | Blue/red/yellow
|
✝ | 3 | 4 | 3 | 12 | A51845 |
Blue/violet/yellow
|
4 | 3 | 4 | ✝ | 13 | A51846 | |
Blue/red/violet
|
4 | 4 | ✝ | 3 | 13 | A51844 | |
Blue/red/violet 6
|
6 | 3 | ✝ | 3 | 14 | A51847 | |
4 | Blue/red/yellow/violet
|
4 | 3 | 4 | 3 | 16 | A51848 |
Blue/red/yellow/violet 6
|
6 | 2 | 3 | 3 | 16 | A51849 | |
*Number of detection channels includes all fluorescence channels as well as a forward scatter and a side scatter channel. ✝Laser upgrade available; green laser not available on Attune CytPix |
The Attune CytPix flow cytometer is 9 cm (3 in) taller than the Attune NxT flow cytometer due to the addition of the high-speed brightfield camera, preserving its small footprint and biosafety hood compatibility, thus saving bench space.
Optics: Fluorescence detection |
Laser power |
Laser |
Wavelength (nm) |
Beam-shaping optics (BSO)* (mW) |
Diode power** (mW) |
Violet |
405 |
50 |
100 |
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Blue |
488 |
50 |
100 |
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Yellow |
561 |
50 |
100 |
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Red |
637 |
100 |
140 |
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* Amount of measured usable laser power after light has gone through the beam optics and shaping filters. ** Vendor-specified theoretical maximum. |
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Laser excitation |
Optimized excitation for minimized stray laser-line noise and losses to reflection |
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Laser profile |
10 x 50 μm flat-top laser providing robust alignment |
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Emission filters |
Up to 14 color channels with wavelength-tuned photomultiplier tubes (PMTs); user-changeable, keyed filters |
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Laser separation |
100 μm |
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Optical alignment |
Fixed alignment with prealigned welded fiber; no user maintenance required |
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Onboard thermoelectric cooler |
No warm-up delay; fiber isn’t affected by “on/off” |
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Simmer mode |
Instant “on/off” reduces usage and/or aging by 10x; only turns on when acquiring samples; reports hours of usage |
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Flat-top laser specified at the flow cell |
Coefficient of variation (CV) <3% over the width of the flat-top laser |
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Upgradable |
Convenient field changes |
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Optics: Imaging |
Laser excitation |
405 nm |
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Pulse width |
< 50 nanoseconds |
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Fluidics |
Flow cell |
Quartz cuvette gel coupled to 1.2 numerical aperture (NA) collection lens, 200 x 200 μm |
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Sample analysis volume |
20 μL–4 mL |
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Custom sample flow rates |
12.5–1,000 μL/min |
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Sample delivery |
Positive-displacement syringe pump for volumetric analysis |
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Sample tubes |
Accommodates tubes from 17 x 100 mm to 8.5 x 45 mm |
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Fluid-level sensing |
Active |
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Standard fluid reservoirs |
1.8 L focusing fluid tank, 1.8 L waste tank, 175 mL shutdown solution tank, and 175 mL wash solution tank |
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Fluid storage |
All fluids stored within instrument |
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Extended fluidics option |
Configuration for 10 L fluid |
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Nominal fluid consumption |
1.8 L/day |
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Automated maintenance cycles |
≤15 min start-up and shutdown—deep clean, sanitize, and debubble modes |
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Performance: Fluorescence detection |
Fluorescence sensitivity |
≤80 molecules of equivalent soluble fluorochrome (MESF) for FITC, ≤30 MESF for PE, ≤70 MESF for APC |
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Fluorescence resolution |
CV <3% for the singlet peak of propidium iodide–stained chicken erythrocyte nuclei (CEN) |
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Data acquisition rate |
Up to 35,000 events/sec, 34 parameters, based on a 10% coincidence rate per Poisson statistics |
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Maximum electronic speed |
65,000 events/sec with all parameters |
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Carryover |
Single-tube format: <1% |
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Forward and side scatter sensitivity |
Able to discriminate platelets from noise |
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Forward and side scatter resolution |
Optimized to resolve lymphocytes, monocytes, and granulocytes in lysed whole blood |
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Forward scatter |
Photodiode detector with 488/10 nm bandpass filter |
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Side scatter |
PMT with default 488/10 nm bandpass filter; optional 405/10+OD2 nm bandpass filter |
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Fluorescence detectors |
14 individual detectors |
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Electronic pulse |
Measured area, height and width pulse for all detectors |
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Violet side scatter resolution |
Can be configured for violet side scatter to better resolve particles from noise |
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Minimum particle size |
0.2 μm on side scatter using submicron bead calibration kit from Bangs Laboratories—0.1 μm on side scatter under following conditions: Using an Attune NxT Flow Cytometer with standard 0.5 mm blocking configuration, an Invitrogen Attune NxT 488/10 Filter (Cat. No. 100083194), and Attune Focusing Fluid (Cat. No. 4488621, 4449791, or A24904) that has been passed through a 0.025 µm filter |
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Performance: Imaging |
Pixel Resolution |
0.3 microns/pixel |
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Objective Magnification |
20x |
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Objective Numerical Aperture (NA) |
0.45 |
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Theoretical Resolution |
0.6 micron |
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Detection Limit |
Visually detect 800 nanometer particles |
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Image Capture Rate |
Up to 6,000 images/second depending on image size and event rate |
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Image Size |
96 x 96 pixels–248 x 248 pixels |
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Field of View |
29 x 29 μm–74 x 74 µm |
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Software |
Compensation |
Full matrix—automated and manual modes, on-plot compensation tools for fine adjustment; use of tubes and wells |
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Flow rate |
Precise flow rate control via software; no hardware adjustments |
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Live streaming |
Live update of statistics during acquisition of events up to 35,000 events/sec |
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Overlays |
Comparative analysis between samples; 3D view |
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Sample recovery |
System able to return unused samples |
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Concentration |
Direct concentration measurement without use of counting beads |
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Software layout |
Fully customizable for each user account |
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Bubble detection technology |
Stops automated run to preserve sample integrity |
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Maximum single-event file |
20 million with option to append |
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Heat map |
Set up for definition of plate layout; screening view for analysis for tubes and plates |
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Threshold |
Up to 4 individual thresholds with user option to apply Boolean logic |
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Gating |
Hierarchical gating with the ability to derive gates |
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Voltage |
User adjustable |
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Window extensions |
User adjustable |
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Area scaling factor (ASF) |
User adjustable |
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Acquisition settings |
Documented in FCS files and maintained upon import |
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Templates |
Create from existing experiments—instrument settings, workspaces, run protocols, heat map settings, and compensation settings optimized and defined previously |
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Tube-to-plate conversion |
One-click transition from tubes to plates and vice versa; no disassembly, no additional QC, no reboot required for conversion between plates and tubes |
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Graphics resolution |
Publication-quality data plots: support for TIF, PNG, BMP, JPG, GIF, and EMF; quickly copy and paste data plots to any external application (e.g., Microsoft™ PowerPoint™ software) |
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User account administration |
Administrative creation of individual user accounts with designated roles, advanced setting permissions, management of individual accounts, user time tracking, and sample count |
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Imaging software |
Image Capture settings |
Set total number of recorded images, image frequency, image capture gate, image size and position, focus, and illumination for control over experiment design and data footprint |
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Image view |
Image view option allows overview of the gallery of images combined with Cell image option to view individual images on the workspace for any cell population |
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Image backgating |
Correlate images to flow cytometry data by backgating either all or selected images onto supported workspace plots |
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Image Measurement tool |
Elliptical tool to measure the area of events in images in µm2 |
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Image Export options |
Export images as 8-bit TIF, PNG, GIF, BMP, JPG, or EMF |
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Image analysis |
Image analysis models |
leukocytes; beads |
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Parameters /features |
>25 measured image parameters in 5 categories: shape features, intensity features, object features, pixel features, and system features |
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Sample size range of models |
5-20 μm |
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Image processing speed |
Fast: 1,000 images/second |
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Pixels |
16-bit image (248 x 248) |
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File type |
extended FCS |
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File type |
<10% error on shape features |
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Quality and regulatory |
Instrument tracking |
Automated daily baseline and performance test with Levey-Jennings plots |
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Warranty |
1 year |
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Production verification testing |
Each instrument is tested and verified for assembly integrity and performance to specifications |
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Quality management system |
Manufacturing standards comply with the requirements of ISO 13485:2003 |
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Robust installation specifications |
Units installed by engineer; preplanning checklist, delivery, and installation; and performance validation compliance with standardized procedure |
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Regulatory status |
For Research Use Only |
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Computer |
Software requirements |
Invitrogen Attune Cytometric Software |
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Monitor |
27 in. flat panel (1,920 x 1,080 resolution); dual-monitor capability |
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Computer |
Minitower desktop |
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Operating system |
Microsoft™ Windows™ 10 64-bit |
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FCS format |
FCS 3.1, 3.0 |
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Processor |
Intel™ Core™ i7 processor |
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RAM |
64 GB |
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Hard drives |
2 x 8 TB SSD, 560 MB/sec; controller RAID1, integrated |
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GPU |
NVIDIA Quadro P2200 |
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Installation requirements |
Electrical requirements |
100–240 VAC, 50/60 Hz, <150 W |
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Heat dissipation |
<150 W |
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Temperature operating ranges |
15–30°C (59–86°F) |
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Operating humidity |
10–80%, noncondensing |
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Audible noise |
<65 dBA at 1.0 m |
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Instrument size |
~49 x 58 x 43 cm (19 x 23 x 17 in.), including fluid bottles |
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Weight |
~33 kg (73 lb) |
For Research Use Only, not intended for use in diagnostic procedures.