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

Getting A New Flow Cytometer? Try Before You Buy (And 2 Other Tips)

Getting A New Flow Cytometer? Try Before You Buy (And 2 Other Tips)

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

5-Point Guide To Buying A New Microscope For Your Lab

5-Point Guide To Buying A New Microscope For Your Lab

By: Heather Brown-Harding, PhD

Have you ever noticed how painful it can be to purchase a new microscope? It would be hard to miss – this can be a frustrating process. A lot of scientists and students consider the new microscope hunt quite scary for a variety of reasons. It might be that you’re worried you won’t get the right microscope and that you’ll regret it, or you may find that dealing with salespeople, in general, makes you kind of uncomfortable. But remember, salespeople are just human beings like you and me, and if we can treat them as such, the whole process of…

Ask These 7 Questions Before Purchasing A Flow Cytometer

Ask These 7 Questions Before Purchasing A Flow Cytometer

By: Tim Bushnell, PhD

I am still convinced that my first cell sorter was possessed. The number of issues that I had with the system remains hard for me to believe, even after all these years. It had been purchased, in part, from one vendor because the sales rep for a competitor was nowhere to be found. At that time, I admit I wasn’t overly diligent in my research process. Since then, I’ve pinpointed some critical questions that need to be answered before purchasing a new instrument. At the end of the process, a shiny new instrument will arrive at your facility. Make sure…

Instrument Quality Control For Reproducible Flow Cytometry Experiments

Instrument Quality Control For Reproducible Flow Cytometry Experiments

By: Tim Bushnell, PhD

The flow cytometer is an integral component of any flow cytometry experiment, and special attention should be paid to ensuring that it is working correctly and consistently. As an end-user, the researcher should be able to sit down at a machine and know that it is performing the same way today as it was yesterday and last week. Equally important is that if any changes in instrument performance have occured, the end-user knows how they have been addressed and corrected, rather than letting them fester and potentially affect the results. Quality control measurements can include a variety of targets, such…

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

By: Tim Bushnell, PhD

Preparing for rare event analysis requires an understanding of the power and limitation of the instrument to be used. From how fast to run the fluidics, to how the signal is processed to the number of gates that can be used in the sorting experiment, each factor impacts the outcome of the experiment.

3 Advantages Of Using The ZE5 Cell Analyzer

3 Advantages Of Using The ZE5 Cell Analyzer

By: Tim Bushnell, PhD

Since the first laser was mounted to create the first flow cytometer, there has been a push for more - more lasers, more detectors, more colors. As a result, today’s researchers require a large number of lasers and detectors to ensure current panels can be run and new, expanded panels can be developed. This can be problematic because, in general, making one decision to improve a cell analyzer can limit the analyzer in other ways. It may seem like an impossible task, but the team of Bio-Rad and Propel Laboratories, collaborated to bring the ZE5™ Cell Analyzer to the market…

3 Advantages FCS Express 6 Has Over Other Flow Cytometry Data Analysis Software Programs

3 Advantages FCS Express 6 Has Over Other Flow Cytometry Data Analysis Software Programs

By: Tim Bushnell, PhD

FCS Express is the ideal data analysis software program to use when analyzing your flow cytometry experiments because it is the most user-friendly program available that is both aligned with current data analysis best practices and maintains rigorous quality control standards.

How To Use A Threshold To Reduce Background Noise In Flow Cytometry

How To Use A Threshold To Reduce Background Noise In Flow Cytometry

By: Tim Bushnell, PhD

Getting a clear signal with reduced noise is an essential component to good data. Adding a threshold when acquiring flow cytometry data is one way to do that. It reduces the number of events by setting a bar that a signal pulse must clear before it is counted as an event. Depending on the importance of the data, the downstream applications for the data (or sorted cells) will dictate how critical the threshold is. In combination with proper sample preparation, appropriate thresholding will reduce debris and ensure best outcome.

How To Set And Monitor Optimal Voltages For A Flow Cytometry Experiment

How To Set And Monitor Optimal Voltages For A Flow Cytometry Experiment

By: Tim Bushnell, PhD

The best way to take out the fear and agony of setting voltages is to use some optimization methods. The peak 2 method is a useful and robust method of identifying optimal PMT voltage ranges. Refining that to the voltage walk with the actual cells and fluorochromes of interest will further improve sensitivity, which is especially critical for rare cell populations or emergent antigens. This article describes how to set up, monitor, and maintain optimal voltage settings for your flow cytometry experiment.

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.