3 Ways The ZE5 Cell Analyzer Accelerates Flow Cytometry Research Opportunities

As new instruments come on the market, vendors are quick to provide data proving the systems’ prowess including sensitivity, speed, and such. These are important characteristics of the instrument, and should be reviewed. However, the real questions that should be asked about any new instrument should look beyond these benchmarks. Specifically, the questions that often come to mind include:

  1. Will the new instrument improve current experimental workflows?
  2. Will the new instrument enable new and novel experimental questions?
  3. Will the new instrument help improve the reproducibility of experiments?

Evaluating the instrument in the context of these questions will help determine if acquiring the instrument will expand the capabilities for the local research community. In the case of the ZE5 Cell Analyzer, it is clear that with the advancements that have been made by the Propel and Bio-Rad teams, this instrument offers significant expansion of capacity, resulting in improved reproducibility of the data.

Several features of the ZE5 stand out as prime examples of why this new instrument is a must-have for the research lab.

  1. Improve Reproducibility — A “Flying Collar Wash Station’” on the ZE5 is designed to wash the sample probe between samples to reduce carryover. For years, researchers have had to manually wash the SIP between samples, to help reduce sample-to-sample carryover. Automating this feature is a huge benefit of the ZE5.By automating the process of cleaning the SIP, carryover is reduced. This in turn reduces one source of data variation. This is even more critical when considering rare event analysis, where sample carryover can potentially skew the data. The data below shows how efficient this system is.

Figure 1: Carryover between samples on ZE5: (A) Lysed whole blood was run on the ZE5 in high-throughput (HT) mode. After each sample, the system carried out an automatic wash cycle of 0.25 sec. outside and 1.75 sec. inside the SIP. A clean tube of water was run immediately after the wash to evaluate carryover. (B) The resulting carryover data showing an average carryover of 0.046% (+/- 0.023%).

  1. Five-laser, 27 fluorescent parameter — More lasers and detectors is an excellent feature and offers improvement for standard assays, enables new assays, and can be used to improve reproducibility of experiments. More detectors allow for a deeper characteristic of a given population. In the case of a hard-to-obtain sample, more detectors allows the researcher to have a larger breadth of characterization, so that the critical data can be obtained without having to split the sample, thus reducing the sensitivity of measurement.With a large number of detectors, the ZE5 can also enable improved labeling of cells by allowing the researcher to ‘“barcode” their samples. In fluorescent barcoding experiments, each sample is labeled with a combination of 2 or more fluorochromes at one of several concentrations. For example, if one uses 2 different fluorochromes, with 3 different intensities (low, medium, and high), it is possible to mix 6 different samples together. A 3 by 3 barcoding results in 9 samples.All the samples are mixed together before they are labeled with the antibody mix at the same time, under the exact same conditions. This improves the staining, and thus the reproducibility of your data, and with the added speed the ZE5 has for sample acquisition (see below), barcoded samples can be read in the same time as a single sample on a slower instrument.If you are interested, you can read about fluorescent barcoding in these papers by Krutzik and Nolan, and Krutzik et al.
  2. Superfast electronics — The fluidics of the ZE5 can deliver a stable flow rate up to 2.5 μl/second. However, without matching fast processing electronics, the speed (and sample) would be wasted with increased coincident events and a high abort rate. The ZE5 delivers in the speed category, with very fast electronic and a cell laser transit time that is 3x as fast as other systems on the market.

Figure 2: Stability of Signal at high acquisition rate: Beads were acquired at increasing events per second, and singlet beads were gated using pulse geometry gating. The %CV of two parameters (Side Scatter and FITC) were plotted over a range from approximately 4,000 eps to 129,000 eps. The mean and standard deviation of the CVs over this range are shown below.

As can be seen, the electronics are stable, with a tight CV shown through a wide speed range. So, in addition to the barcoding discussed above, the fast electronics and stable flow rates are enabling for rare event analysis. Imagine trying to measure a cell that is found at a frequency of 1 in 105 cells. With rare events like this, the statistics are governed by Poisson distributions, rather than the more familiar Gaussian distributions. In Poisson statistics, it is the number of positive events that is important, not the total number of events.

Figure 3: Time to collect 400 positive events. The time to collect 400 events of a rare population (1 in 105 cells) is plotted versus the speed of acquisition (in events per second).

As this figure shows, to collect 40 million events with a typical flow cytometer is going to take 150 minutes (2.5 hours) for a single sample. However, with the speed of the ZE5, these rare event experiments become possible, as even at a moderately fast rate of 60,000 events per second, collection time drops to less than 12 minutes. Thus, the ZE5 enables researchers to study and characterize rarer cell populations in a reasonable time.

Some technological advances are incremental, while others are significant game-changing tools that offer the researcher the ability to significantly improve current assays while allowing for new and novel avenues of research to be performed. With speed, sensitivity, and capacity to spare, the ZE5 fits into the game-changing category. Reduced carryover, increased speed of acquisition, and a large number of parameters all open up new and novel assays, while improving the quality and reproducibility of ongoing ones.

To learn more about 3 Ways The ZE5 Cell Analyzer Accelerates Flow Cytometry Research Opportunities, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Join Expert Cytometry's Mastery Class
Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

Similar Articles

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

By: Tim Bushnell, PhD

What photon from yonder fluorochrome breaks?  It is … umm… hmmm. Let me see. Excitation off a 561 nm laser, with an emission maximum of 692 nm. I’m sure if Shakespeare was a flow cytometrist, he might have written that very scene. But the play is lost in time. However, since the protagonist had difficulty determining what fluorochrome was emitting photons, let’s consider how this could be figured out. In my opinion, one of the handiest flow cytometry tools is the spectral viewer. This tool helps visualize the excitation and emission profile of different fluorochromes, as well as allowing you…

3 Must-Have High-Dimensional Flow Cytometry Controls

3 Must-Have High-Dimensional Flow Cytometry Controls

By: Tim Bushnell, PhD

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

By: Tim Bushnell, PhD

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

Flow Cytometry Year in Review: Key Changes To Know

Flow Cytometry Year in Review: Key Changes To Know

By: Meerambika Mishra

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

What Star Trek Taught Me About Flow Cytometry

What Star Trek Taught Me About Flow Cytometry

By: Tim Bushnell, PhD

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

5 Flow Cytometry Strategies That Sun Tzu Taught Me

5 Flow Cytometry Strategies That Sun Tzu Taught Me

By: Tim Bushnell, PhD

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

By: Meerambika Mishra

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest.  Cells Flow cytometry measures biological processes at the whole cell level. To do…

Which Fluorophores To Use For Your Microscopy Experiment

Which Fluorophores To Use For Your Microscopy Experiment

By: Heather Brown-Harding, PhD

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

4 No Cost Ways To Improve Your Microscopy Image Quality

4 No Cost Ways To Improve Your Microscopy Image Quality

By: Heather Brown-Harding, PhD

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.