Cytometry and Antibody Technology

Attune NxT

Traditional benchtop analyzer
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Overview

The Attune NxT is the only platform that utilize acoustic focusing. This allows the sample to be processed at a much higher pressure without a significant impact on data quality.

Features include:

  • 4 lasers (405nm, 488nm, 561nm and 640nm) and 14 detectors
  • A plate loader – handles 96 and 384 well plate of any type
  • FSC threshold of detection of 200nm
  • Intuitive software – similar to BD FACS DIVA and easy to master
  • Acoustic focusing – forces the samples in line through sound waves
  • Volumetric acquisition – provides true count of the cells in each sample
  • No restriction in the type of sample tube that can be used

Acoustic Focusing

The Attune is equipped with a piezo-electric device upstream of the point where the sample reach the stream of PBS. It emits sound waves that focuses the cells in a single line.

This way every event gets the best possible illumination in front of the laser beam, regardless of the pressure applied to the sample. The video on the left, taken from the ThermoFisher website, illustrates the process.

Sample Preparation Tips

Accepted Tubes

The Attune accepts 5mL round bottom tubes (FACS tubes), 1.2mL round bottom tubes (“bullet tubes”), and 1.7mL Eppendorf tubes. The plate loader accepts a variety of 96- and 384- well plates including deep-well.

Sample Volume/Concentration

To avoid getting a very high number of aborted events (due to cells crossing the laser at the same time) follow the guide below to decide on an appropriate sample pressure.

Resources

  • Attune NxT Quick Guide
  • Having problems viewing group names from Attune FCS files in FlowJo? This guide will show you how to fix that.

There is also a lot of information available in the Resources Library (located under the Resources menu)! The ones relevant to the Attune can also be found in the module below. Recommended voltages specific the Attune can be found on the “Setting Voltages” page.

 

Configuration and Suggested Fluorophores

When designing a panel, only one fluorophore per row should be used – it is not possible to distinguish fluorophores/dyes from others within in that same row. This table is also posted near the physical cytometer to refer to the detectors used to identify each fluorophore. This table do not include a complete list of all fluorophores, only those that are commonly used.

Troubleshooting

Section under construction, updates coming soon!
Instrument Not Connecting

Try a hard reboot:

  1. turn off the software first,
  2. then the Attune NxT cytometer
  3. then the Autosampler

Turn on in reverse, allowing 30 seconds between each step (i.e., turn on the Autosampler, wait 30 seconds, turn on the Attune NxT cytometer, wait another 30 seconds, and then turn on the software). Failure to wait an appropriate amount of time may result in a connection error.

Windows Is Telling Me to Reset my CNET Password

Contact the staff. This is an issue with the Attune computer; you do not need to change your CNET password.

Leak Detector Triggered, but There is No Leak

If the leak detector got in contact with water or sheath, it will prevent usage of the instrument until the moistures removed. The hair dryer is in the drawer under the Attune NxT desk.

 

Shutdown and Maintenance
  • If you are the last user of the day, run the Standard Shutdown from the Instrument tab and follow the prompts.
    • If it is a Friday, initiate the Thorough Shutdown from the Shutdown drop-down menu. 
    • Once Shutdown is initiated (bleach is loaded) you can close the software, log out of Windows, and walk away.
  • If there are subsequent users, run the Sanitize SIP script from the Instrument tab.