Attune™ NxT Flow Cytometer, blue/red/violet/yellow - FAQs

To minimize the number of failed performance tests on your Attune NxT Flow Cytometer, we suggest the following:

•   Make sure to carry out daily, weekly, and monthly maintenance procedures (pages 8-14 of the Maintenance and Troubleshooting Guide, linked below).
•   Always run 3 startup cycles and 2 Rinse procedures (pages 46-47 of the Maintenance and Troubleshooting Guide linked below).
•   If the performance test fails, carry out the following priming sequence: run 3 startup cycles, 2 de-bubble cycles, and 5 rinse procedures, followed by running the SIP sanitize function.

Please review the Attune Flow Cytometry Maintenance and Troubleshooting Guide for more information about performing routine maintenance procedures and troubleshooting.

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Yes. The Attune Nxt Flow Cytometer can detect down to 0.2 µm and using a special configuration, down to 0.1 µm. Optimize your settings using Flow Cytometry Sub-micron Particle Size Reference Kit, Cat. No. F13839.

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Yes. You would need to purchase Attune NxT Custom Filter Holder Kit, Cat. No. A27784 to make your own custom emission or dichroic filters in order to customize the optical configuration of the instrument. Each kit contains sufficient parts to make 2 emission filters and 2 dichroic filters. The kit includes instructions for attaching the filter to the filter blade, but does not include the glass filter. Other accessories to optimize for special applications include Cat No. 100083194 Attune NxT Small Particle Side-Scatter Filter, Cat. No. 100083194 and Attune NxT Blocker Bar Conversion Kit, Cat No. A35966.

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Yes. There are 6 lasers to choose from (blue, green, yellow, red, violet, and violet 6). You can start with one laser and at any time upgrade the same instrument with up to 4 lasers to utilize a maximum of 14 detection channels. Using the Violet 6 laser along with the blue, red, and yellow lasers, one can detect up to 16 channels.

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Because of the acoustic-assisted hydrodynamic technology, the Attune NxT Flow Cytometer uses only 1/10th the amount of fluids, and is 10X faster and less sensitive to clogging compared to standard flow cytometers. You can set the flow rate from 12.5-1,000 µL/min and collect up to 65,000 events/sec using from 20 µL to 4 mL of sample volume. These unique properties allow for rare event analysis, as well standard flow analysis.

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Filters are recommended for replacement once every three months, unless performing small particle detection when once-a-month replacement is recommended.

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In an emergency, we recommend using the highest purity of water available in place of the shutdown solution and then switching to the Attune Shutdown Solution as soon as possible.

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In an emergency, we recommend using the highest purity of water available in place of the wash solution and then switching to the Attune Wash Solution as soon as possible.

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You can tell whether the compensation data are included by looking for the $SPILL or $COMP keyword embedded within the FCS files. You can also see the FCS file information within the Attune NxT software by going to the "View" tab and checking the box labeled "FCS File Panel". This will open a window which shows the information contained in the FCS file associated with the selected sample. It will include any compensation ($SPILLOVER) that has been done.

Step 2 allows for antibodies you have already purchased or are from a vendor outside Thermo Fisher Scientific. If you are undecided about which format you want for something, you can add the antigen name in the "Antibodies you need" section and then choose a placeholder fluorochrome in the following step. Both will help you keep track of channels that are reserved for reagents that you do not plan to buy from Thermo Fisher Scientific.

There are both PDF (printable) and CSV (spreadsheet) exports available in the summary step for you to take away for reference and ordering purposes.

Yes, you can define a new cytometer using the "Enter your cytometer manually" link below the cytometer dropdown menu.

Select a cytometer from the dropdown menu and then select the "Edit cytometer settings" link to modify the configuration as needed.

Each dot indicates a different fluorochrome that Thermo Fisher Scientific has available in each channel of the cytometer you have chosen.

You may export the spreadsheet found on step 5 and share it.

Select your target species first, and then type your antigen. A drop-down list will appear. Select the antigen from the list and click on "Confirm selections." If you have an antibody that is not on the list, in the "Antibodies you already have" section, you can enter a name and select "use this name anyway."

Yes, the previous work is saved. If you plan to leave the Panel Builder and come back later, be sure to select "Save" in the lower right corner to save your panel.

No. Many users are using unstained cells in combination with FMO controls to identify their positive populations.

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There are several things that can cause a low event rate:

-The system may be clogged
-The threshold level may be set too high
-The PMT voltage for the threshold parameter may be set too low
-The sample may be too dilute or not adequately mixed
-The sample syringe may be loose

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

There are several things that can cause a high event rate:

-There may be an air bubble in the flow cell
-The threshold level may be set too low
-The PMT voltage for the threshold parameter may be set too high
-The sample may be too concentrated
-The sample flow rate may be too high
-The sample may have a bacterial contamination

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Open the Attune NxT software, open the main menu, and go to the “Instrument” tab. The option on the far left is “System Log”. Click on that and it will open the log file. At the bottom of the page there is a button labeled “Export”. Use this to export and save the log file.

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We recommend looking inside the box, possibly underneath the bag of buffer.

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Send an email to Cellular Analysis Technical Support at technical-molecularprobes@lifetech.com, including the words “flow*#148; or “Attune” in the subject line for proper routing of your email. Please describe the type of application you are doing, how you set up the experiment, and your results. It would be ideal if you could send us a sample of your data that we can review.

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If the issue is clearly an instrument problem, send an email to Instrument Services at instrumentservices@thermofisher.com with your instrument serial number. If the issue is software or application-related, or if you do not know the source of the problem, send an email to Cellular Analysis Technical Support at technical-molecularprobes@lifetech.com, including the words “flow*#148; or “Attune” in the subject line for proper routing of your email.

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For help with designing panels for flow cytometry, see our new Molecular Probes Flow Cytometry Panel Design Tool (http://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-panel-design-tool.html) The panel design tool can help you choose fluorescent antibody conjugates for your flow cytometry panel in a few easy steps: pick the antibody species reactivity, select up to 14 targets of interest (choices include viability dyes), and choose the lasers or fluorophores you want to view.

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It depends on your instrument. People have done 17 colors (Perfetto SP, Chattopadhyay PK, and Roederer M (2004) Seventeen-colour flow cytometry: unraveling the immune system. Nat Rev Immunol 4:648–655 (http://www.ncbi.nlm.nih.gov/pubmed/15286731)) and more, but it takes a lot of planning and testing to make it work. The Attune NxT Acoustic Focusing Cytometer can run up to 14 colors plus forward and side scatter with a system equipped with all four lasers (violet, blue, yellow and red).

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An FMO control (flow minus one) is a control in which you label cells or beads with every color in your panel, omitting one. Make one FMO control for each color in your panel. These controls are important for helping you properly set gates on your data.

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The flow cytometry field is moving away from using isotype controls as they are not necessarily the most appropriate way to control for non-specific binding. Instead, they are using unstained cells to define the negative population, single stained controls to set compensation, and flow minus one (FMO) controls to set regions and gates.

You may want to consider whether using an isotype control is something you need to do. Here are some references you might want to look at:
-O'Gorman MR, Thomas J. (1999) Isotype controls-time to let go? Cytometry 38:78-80.

-Keeney M, Gratama JW, Chin-Yee IH et al. (1998) Isotype controls in the analysis of lymphocytes and CD34+ stem/progenitor cells by flow cytometry- time to let go! Cytometry 34:280-283.

-Hulspas R, O'Gorman MR, Wood BL et al. (2009) Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin Cytom 76:355–364.

-Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline - Second Edition. Clinical and Laboratory Standards Institute, (CLSI). Document H42-A2 Volume 27 No.16, 2007.

-Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline - Second Edition. Clinical and Laboratory Standards Institute, (CLSI). Document H43-A2 Volume 27 No. 11, 2007.

If you do wish to use isotype controls, we offer a wide variety of species and fluorophores, search our Web site under "isotype antibody control."

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Our Flow Cytometry Size Calibration Kit has non-fluorescent particle-size calibration standards that provide a simple, accurate way to determine cell sizes by flow cytometry. The kit contains six suspensions of highly uniform polystyrene microspheres with the following diameters: 1.0 µm, 2.0 µm, 4.0 µm, 6.0 µm, 10.0 µm and 15.0 µm. All of the microsphere suspensions are provided in convenient dropper vials. This kit looks at the scatter properties of non-fluorescent microspheres of known sizes to determine approximate cell sizes.

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The smallest size that you can detect with the Attune NxT Acoustic Focusing Cytometer is 0.5 µm.

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The Attune NxT Autosampler, an optional accessory for the Attune NxT Acoustic Focusing Cytometer, enables rapid processing of up to 384 samples. It has broad compatibility with different plate formats, both 96- and 384-well plates. It has an intelligent probe designed to minimize clogging and carryover (<0.5%) and to prevent damage to the instrument. It mixes by aspiration rather than shaking to ensure homogeneity of the sample and maintain cell viability. Is performs automated cleaning as part of the shutdown process of the Attune NxT Cytometer. It provides consistent data regardless of sampling method (tube vs. plate) and collection rate.

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-Modular design - Multiple configurations available - field upgradable.
-Save time - 10X faster speeds with no loss in data quality.
-Simplified sample prep - No wash, no lyse options, non-clogging fluidics.
-Enables unique applications - Complex protocols on a broad range of cell types and samples.

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With the option to be configured with up to 4 lasers and 14 colors for multi-parameter analysis the Attune NxT Acoustic Focusing Cytometer was designed as a modular system to fit most experimental needs and lab budgets. The novel design of the optical path helps ensure precise fixed alignment of four spatially separated lasers onto the sample stream enabling consistency in data over time, superior performance, and superior reliability. The instrument can be configured with up to 4 solid-state lasers (405 nm, 488 nm, 561 nm, and 637 nm) with flat top beam profiles.

The Attune NxT Flow Cytometer's acoustic focusing uses ultrasonic radiation pressure (> 2 MHz) to transport particles into the center of the sample stream. This pre-focused stream is then injected into the sheath stream, which supplies an additional hydrodynamic pressure to the sample. The combination of these two forces- termed acoustic-assisted hydrodynamic focusing-results in a narrow core stream and uniform laser illumination, regardless of the sample input rate. In traditional cytometers that rely solely on hydrodynamic focusing, the sample core widens to accommodate the increases in flow rate, which results in less uniform laser light illumination.

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Cytometry is the measurement of physical or chemical characteristics of cells or particles. Flow cytometry measures these characteristics of cells or particles as they individually pass lasers in a flow cytometer instrument. Flow cytometry is performed on single cells, providing discrete measurements for each cell in the sample. It also provides a statistical distribution of the measured characteristics of the sample.

A flow cytometer is made up of three subsystems: fluidics, optics, and electronics. Fluidics moves the cells and introduces them for interrogation. Optics generates and collects the light signals. Electronics converts the optical signals to proportional electronic signals for computer analysis.

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