Customer Experiences with the Attune NxT Flow Cytometer

Reading about fellow researchers' experiences and consulting with colleagues for direct feedback on their hands-on experiences with instruments are useful when considering the purchase of a flow cytometer, especially if you speak with labs working with similar sample types, throughput volume, or applications. Many researchers will gladly discuss how they reasoned through their instrument decision, what they would have done differently, and what they like and dislike about their flow cytometer after having it up and running in their lab.

Read about how a few Attune NxT Flow Cytometer customers rate the instrument across several variables:

Precision

Takashi Satoh

“When all is said and done, it outperforms [the rest] in that it can analyze 35,000 cells per second with acoustic focusing technology. Its accuracy is not diminished even if you conduct analysis at a high flow rate of 100–1,000 µL/min, which drastically shortens the time required for an experiment.”
Dr. Takashi Satoh, IFReC Assistant Professor, Osaka University, Japan

Tim Inglis

“We’ve been very comfortable working with Thermo Fisher Scientific for the last four years. We have a collaboration arrangement that goes back over three and a half years and has taken us from initial, in some cases, accidental/ serendipitous discoveries, to a point where the technology we’re working on is being adopted by internationally respected reference laboratories overseas. That’s very unusual for a group based in somewhere as geographically isolated as Western Australia. This collaboration is a very unusual one, at least, for us locally and has been a very productive one.”
Tim Inglis, BM, DM, PhD, FRCPath, FRCPA, DTM&H, School of Medicine, University of Western Australia, PathWest Laboratory Medicine

Mike Meyer

“Overall, the Attune Nxt is a powerful flow cytometer with a nice, small footprint. The software is easy to learn and easy to use. The system can be used for a variety of applications; just right for a multi-user shared facility.”
Mike Meyer, Cytometry Facility Manager, Hillman Cancer Center, University of Pittsburgh

Jordi Petriz

"I knew I had learned a lot during 25 years of experience doing research with flow cytometry. Now I am surprised to see how much I can learn doing research with the Attune NxT, and how this new technology can be very helpful to make visible the invisible.”
Jordi Petriz, PhD, Josep Carreras Leukemia Research Institute, Barcelona, Spain, Councilor at ESCCA Executive Board, Past-President of the Iberian Society for Cytometry

Kieran Mulroney

“We are looking for the effects of the antibiotic drug on individual cells in a very rapid manner. Traditional antibacterial sensitivity testing can take 1–3 days depending on how difficult the bacteria are to grow, isolate, and then test for sensitivity. The Attune NxT allows us, within 1 hour, to tell whether or not the drug will effectively treat the infection. Within 3 hours, we can accurately assay the minimum drug concentration needed to inhibit bacterial growth.”
Kieran Mulroney, Biomedical Sciences Researcher, Harry Perkins Medical Research Institute, University of Western Australia

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Clogging

Many sample types for flow cytometry have high susceptibility to clogging. How does the Attune NxT Flow Cytometer perform?

Charles Prussak

“We needed to have a flow cytometer that would allow us to take samples from tumors that had tended to clog other machines. Therefore the Attune NxT is the flow cytometer of choice for our application. The Attune NxT has a number of characteristics that are critical for our research program. Not only does it have acoustic focusing, which will allow us to go at higher sample throughput, but it also has a larger flow cell, so when we’re looking at tumor cells and isolating cells from those tumors we don’t have to worry about clogging and having to spend a lot of downtime getting the instrument up and running again.”
Charles Prussak, PharmD, PhD., Director of the Cell Therapy Translational Laboratory (CTTL), University of California San Diego

“One of the problems that everyone is familiar with who works in flow cytometry is clogging. Clogging is a thing of the past with this instrument. You have so many samples that you have to run, you can’t wait between samples to clear everything out.”
Bruno Sainz, CLIP Investigator, Universidad Autónoma de Madrid, Madrid, Spain

How does the Attune NxT Flow Cytometer maintain a high degree of clog-resistance?

The Attune NxT Flow Cytometer features a syringe driven system that works to “PUSH” sample, compared to a max “PULL” on a pressurized system of <15 PSI (see figure).

Pressurized systems pull the sample into the system

  • Max pull on a pressurized system of <15 PSI
  • Prone to fluctuations in viscosity and temperature

Syringe-driven systems push the sample

  • The Attune NxT Flow Cytometer pushes the sample up to 75 PSI
  • Less sensitive to clogs caused by backpressure
  • Robustness to pressure fluctuations
  • Better system diagnosis capabilities

Learn more about the fluidics system of the Attune NxT Flow Cytometer

Pressure PSI measurement comparison of syringe-based system vs. a pressure-driven system

Figure 1. Pressure PSI measurement comparison of syringe-based system vs. a pressure-driven system. (Left) Pressurized systems pull the sample into the system. The maximum pull on a pressurized system is approximately <15 PSI. (Right) The Attune NxT Flow Cytometer employs a syringe-driven system that pushes the sample up to 75 PSI.

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Compensation

As with most multiplex detection experiments, a combination of dyes may have spectral overlap and for optimal simultaneous analysis, compensation is required. Compensation can mean tedious trial-and-error adjustments of compensation matrix coefficients.

J. Paul Robinson

“Phenotyping up to 14 colors is easy with 4 lasers—reduces the number of colors you have to compensate and makes it easier to create assays.”
J. Paul Robinson, PhD, SVM Professor of Cytomics, Professor of Biomedical Engineering, Purdue University

Bruno Sainz

“Multiplexing and compensation are much easier and extremely efficient with the Attune NxT. The Attune NxT is ideal for anyone looking to identify very rare subpopulations of cells with high efficiency and certainty.”
Bruno Sainz, CLIP Investigator, Universidad Autónoma de Madrid, Madrid, Spain

How does the Attune NxT Flow Cytometer address compensation?

Attune software settings

Figure 2. Attune NxT software controls for applying compensation.

The Attune NxT Flow Cytometer is designed to decrease the vulnerability of the compensation process to artifacts and error:

  • Hardware—minimizes color compensation by leveraging 4 spatially separated lasers onto the sample stream, which provide flexibility for experimental setup
  • Software—set of automated and manual modes, on-plot pre- and post-compensation tools for fine adjustment; pre- and post-compensation tools help eliminate tedious trial-and-error and guesswork adjustments of compensation matrix coefficients to increase reliability of the data from tubes and wells
6-panel flow cytometer scatter plots showing compensation

Figure 3. Data with (A-C) and without (D-F) auto-compensation applied. Proper compensation setup is requisite for accurate analysis of and conclusions from complex data sets. The contrast between plots with and without compensation is significant

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Learning curve

Bruno Sainz

“The Attune NxT instrument is easy to use, the software is user friendly and the time from installation to running samples is minimal.”
Bruno Sainz, CLIP Investigator, Universidad Autónoma de Madrid, Madrid, Spain

Interface

Figure 4. Intuitive, user-friendly software with familiar workflow.

Mike Meyer

“One of the more impressive aspects of the Attune NxT is “ease of use”. As a shared facility manager, I instruct a wide variety of users how to run a wide variety of instruments. It is often a challenge to teach a new, and at times, veteran cytometrist how to operate a new system. User friendly software is a must. Several of my facility users picked up on the Attune NxT’s software right away. Many were able to run complex multi-parameter experiments in their first session; some of them were doing this on their own without any assistance from the facility staff. If only all of our instruments were so easy to use!”
Mike Meyer, Cytometry Facility Manager, Hillman Cancer Center, University of Pittsburgh

J. Paul Robinson

“The dual sample tube to plate operation—instant change from tube to plates is really an excellent feature.”
J. Paul Robinson, PhD, SVM Professor of Cytomics, Professor of Biomedical Engineering, Purdue University

Software overview
Figure 5. List of software features.

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Time intensity

James (Jake) W. Jacobberger

“Cytometry is well-suited for quantifying the fractions of cells in cell cycle phases. For the most part, this amounts to measuring DNA content and deconvolving the resulting histogram into G1, S, and G2+M or G1, S, G2+early M, and late M. Considerable effort has been put to coupling DNA content with RNA, protein, or specific epitopes –generally limited to 1-4. In each case, the number of cell cycle compartments increases. In our most recent efforts, using 6 parameters (DNA-area, DNA-peak, Light Scatter, cyclin A2, cyclin B1, and phospho-S10-histone H3), we identify X compartments that are defined objectively by a rule based system.

Fixing and staining were optimized many years ago, and while we have examined many variations, we have not discovered anything noteworthy that improves the analytic quality of the resultant data. Currently, we are working on a “washless” staining assay, relying on a single high dilution to minimize background staining and acoustic focusing to render the sample analyzable over a short time period. The results are striking. The S/N approaches a fully washed sample; the minimized handling results in better cell recovery, and better cell recovery results in better definition of cell cycle compartments with low cell fractions. Additionally, the “washless” assay provides a significant labor savings. We are currently testing this approach with clinical samples of limited cell numbers to detect S phase cells in peripheral blood as part of a pharmacodynamic evaluation of hypomethylating therapy.”
James (Jake) W. Jacobberger, Professor of Mammalian Cell Biology and Cancer Cell Biology, Case Western Reserve University, Cleveland, OH USA

“It greatly improves my quality of life in the lab by shortening the time I have to sit with the cytometer from 2-3 hours with other platforms into 40 minutes with the Attune NxT [instrument].”
Dr. Li-Rung Huang, National Health Research Institutes, Taiwan

J. Paul Robinson

“Having evaluated the Attune NxT over several months, I would say it fits the superior category of flow cytometers. The ability to run VERY dilute samples is quite amazing and might be a life saver on many occasions where you little to no sample left.”
J. Paul Robinson, PhD, SVM Professor of Cytomics, Professor of Biomedical Engineering, Purdue University

Mike Meyer

“The Attune NxT that we evaluated had an Autosampler that also proved easy to use. We looked at several metrics and compared the NxT to other 96 well plate readers. The Autosampler proved to have very good stability and very low carryover. We were most impressed by the way that the Autosampler took advantage of the Attune NxT’s fluidics and high volume thru-put. Without compromising stability or precision, the Autosampler was able to run plates much faster than any other plate reader.”
Mike Meyer, Cytometry Facility Manager, Hillman Cancer Center, University of Pittsburgh

Kieran Mulroney

“We are looking for the effects of the antibiotic drug on individual cells in a very rapid manner. Traditional antibacterial sensitivity testing can take 1 – 3 days depending on how difficult the bacteria are to grow, isolate, and then test for sensitivity. The Attune NxT allows us, within 1 hour, to tell whether or not the drug will effectively treat the infection. Within 3 hours, we can accurately assay the minimum drug concentration needed to inhibit bacterial growth.”
Kieran Mulroney, Biomedical Sciences Researcher, Harry Perkins Medical Research Institute, University of Western Australia

Bruno Sainz

“We wanted to use their technology that is incorporated into the cytometer—this acoustic technology—to identify these very rare populations of cells. It’s doing exactly what we thought it would do: identify these very rare cells, we can see them with very high efficiency and with very small amounts of samples, and at very tremendous fast speeds. We are very used to using flow cytometers where the flow streams are not properly aligned and here we can actually align all of those cells, so it increases our efficiency and ability to identify the cells much better than traditional cytometers that we’ve used before.”
Bruno Sainz, CLIP Investigator, Universidad Autónoma de Madrid, Madrid, Spain

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Price

Takashi Satoh

“Since there is little waste liquid, savings can be made not only on the base price but also on the running costs.”
Dr. Takashi Satoh, IFReC Assistant Professor, Osaka University, Japan

Mike Meyer

“As a shared facility manager, I work with many different assays: whole blood, lyse no-wash, routine cell preps and rare event problems. The Attune NxT is versatile enough to run all of these experiments and powerful enough to generate results comparable to any other multi-laser, multi-parameter cytometer…. just right for a multi-user shared facility. As a bonus, the price is low enough that it could be purchased for use in an individual lab.”
Mike Meyer, Cytometry Facility Manager, Hillman Cancer Center, University of Pittsburgh

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Bench space

Illustration of Attune NxT footprint dimensions

The Attune NxT Flow Cytometer on a desktop workstation in a core facility (shown at the top of this page), weighs 64 pounds and consumes less than 200 watts of power. Wires and optical paths surround components and were designed while maintaining the guiding principles of user access, service access and manufacturing access.

Why does size matter?

Bruno Sainz

“The instrument has a remarkably small footprint, with the fluidics bottles contained within the instrument, making the Attune NxT ideal for any laboratory setting.”
Bruno Sainz, CLIP Investigator, Universidad Autónoma de Madrid, Madrid, Spain

Mike Meyer

“I have had an Attune NxT with Autosampler in my shared facility for the past two years. It is impressive that such a small footprint instrument has all the capabilities of the much larger, more expensive instruments.”
Mike Meyer, Cytometry Facility Manager, Hillman Cancer Center, University of Pittsburgh

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Distinctiveness

Sara De Biasi

“Flow cytometry is fast and precise, and it allows the study of several dozens of proteins expressed at a single cell level. The study of rare cell populations is of growing importance. It’s useful not only to understand disease mechanisms, but also to find novel targets. With multiparameter capabilities and a very high analysis rate, flow cytometry is, at present, the most potent technology to address rare cell analysis. When I first studied iNKT cell populations in HIV-positive patients, I had to deal with a more evident paucity of cells (iNKT cells are rarer in HIV, as HIV-positive people are immunocompromised). More than 20 million cells should be stained to find these rare populations among PBMCs. Recently, no instrument was able to acquire 20 million events. Then the Attune NxT Flow Cytometer made my project reliable. The consequence of acquiring 20 million cells was how data should be managed, but powerful computers with many gigabytes solved this problem.”
Sara De Biasi, PhD, Department of Life Sciences, University of Modena and Reggio Emilia, (UNIMORE), Modena, Italy

David Cousins

"One of the main projects in my laboratory is focused on the description and functional analysis of an emerging innate lymphoid cell type. These cells are extremely rare both in blood and in tissues. For flow cytometry analysis we use the Attune Acoustic Focusing Cytometer. The high sample rates of [the cytometer] allow me to reduce centrifugation steps so that we retain more cells and more rapidly detect rare events. We could not have performed these studies with any other instrument."
Prof. David Cousins, University of Leicester, UK

Bruno Sainz

“Our future directions are to identify circulating tumor cells in blood cells and a methodology I that is incorporated into the Attune NxT Flow Cytometer is a now wash no lyse step. For anyone who has worked with blood samples, we know that we have to first wash our blood samples, lyse the red blood cells, and during this entire process if you are looking for a very rare subpopulation of cells you are going to lose those cells or you are going to lose your efficiency by which you are going to detect them. So just by simply changing out some filters, you don’t have to wash your cells, you don’t have to lyse your cells, you can run your marked cells through the cytometer—essentially run diluted blood through the cytometer and now you can actually see the population with an efficiency that we’ve never been able to achieve before. So I think the NWNL methodology that is incorporated into this cytometer is a benefit for anyone who is doing liquid biopsy assays or looking for populations of cells in blood samples.”
Bruno Sainz, CLIP Investigator, Universidad Autónoma de Madrid, Madrid, Spain

Publication—Guidelines for the use of flow cytometry and cell sorting in immunological studies

Guidelines for the use of flow cytometry and cell sorting in immunological studies, a 214 page publication in the European Journal of Immunology, features a chapter authored by Jordi Petriz and Mike Ward. This special issue covers guidelines for the use of flow cytometry and cell sorting in immunological studies. Included are many of our collaborations and it features a section about the NLNW technique of the Attune NxT Flow Cytometry for Reactive Oxygen Species with minimal sample perturbation authored by Jordi Petriz and Mike Ward.

Title: Guidelines for the use of flow cytometry and cell sorting in immunological studies
Publication: Eur J Immunol, 2017 Oct.; 47(10):1584-1797
DOI: 10.1002/eji.201646632
Citation:   A. Cossarizza, H. Chang, A. Radbruch, M. Akdis, I. Andrä, et. al.

Download the free-access article

Comparison of standard vs. no lyse, no wash workflows
Bruno Sainz

“We wanted to ask a relevant question concerning stem cell biopsy and we focused our project on isolating and identifying circulating tumor cells. Our goal is to identify and to quantify, calculate the number of CTC’s present in patient blood samples. Now based on the technology and the ability of the Attune NxT to identify very small, rare population, we believe we will finally be able to get good reliable data on the number of CTC’s present in patient blood samples. We’ve tried many cytometry platforms and what we have found throughout the years is that it is very difficult to identify the subpopulation of cells. The Attune NxT has acoustic technology which allows you to detect very small, rare population in a sample and sometimes we have milliliters of blood so the speed at which we can run that samples and get the viable data off the number of cells is what we’ve been looking to find for a very long time.”
Bruno Sainz, CLIP Investigator, Universidad Autónoma de Madrid, Madrid, Spain

J. Paul Robinson

“The Attune NxT (instrument) has given us an opportunity to analyze bacteria at a single-cell level. And in combination with the range of vital dyes available to us, it’s expanded the potential repertoire of culture-independent bacteriology well beyond the boundaries that are set by molecular biology and in particular, by real-time PCR.”
Tim Inglis, BM, DM, PhD, FRCPath, FRCPA, DTM&H, School of Medicine, University of Western Australia, PathWest Laboratory Medicine

Steve McClellan

“The focus of our research for the last five years has really been cancer stem cells. These are very rare cells isolated form patient tumors that tend to exhibit a lot of drug resistance. They are very hard to kill in a therapeutic situation. So we study these cells, but with the advent of the exosome technology we want to be able to figure out how these cells communicate with each other and what the particles they release are actually telling the cells around them.

We’ve recently started working with exosomes. The Attune NxT seems to be a very good platform for analyzing exosomes. They’ve developed a new method of ultra-filtering the sheath which reduces background noise to a very low level and gives us confidence that we are actually analyzing individual particles. We think this is going to be a big breakthrough in the field of exosomes.

So after evaluating several platforms, we chose the NxT because of its speed. It is able to analyze cells at a much higher rate, with very good precision, and it allows us to answer questions that other systems have trouble with, especially in the exosome field. The high resolution has been very beneficial in helping us resolve individual exosomes which are extremely small compared to other types of cells. Flow cytometers typically have problems with it.

The field of exosomes has really exploded in the last four years. There are many options of looking at health monitoring, in that we can actually look at patient’s health by looking at exosomes. The bases of this research is that this is how cells communicate with each other, especially in the field of cancer. Tumor cells release exosomes that effect the cells around them and actually make the process of invasion easier. It’s an actionable target for developing potential new cancer therapy. So the field of circulating tumor cells has also really expanded. These are very rare cells—sometimes thousands maybe hundreds in a tube of millions even billions of blood cells. We would normally have to isolate the cells away in other magnetic or other enrichment techniques. The Attune NxT can actually pump cells through so quickly that we can actually use whole blood samples to look and enumerate these very rare cells, which is a unique feature of the Attune NxT.

I believe the future of flow cytometry is very exciting. We have so many new technologies coming online. We see the development of small instrumentation such as the Attune NxT which provides extremely high resolution, multiple colors with multiple lasers, the speed of the sampling which is very unique on this system. We really are very happy with the instrument and believe that it is a good workhorse and fits most all of the applications needs of our core facility.”
Steve McClellan, BS, MT, SCYM (ASCP), Flow Cytometer and Imaging Core Lab Manager at the Mitchell Cancer Institute

Watch the full interview

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