Elucidating Complex Flow Cytometry Studies with theSpeed and Sensitivity of the ZE5 Cell Analyzer
Yasha Talaga, Nico Tuason, and Ma2 Alexander
Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA

Abstract

Recent advancements and innovations in flow cytometry instrumentation have enabled scientists to perform increasingly complex
experiments and to obtain biologically relevant data in a high-throughput manner. Utilizing speed and flexibility, the ZE5 Cell Analyzer
from Bio-Rad Laboratories takes flow cytometry from a technique to a powerful tool that goes beyond instrumentation. With the capability
to sample up to 100,000 events per second and with five lasers with 30 detectors to choose from, the ZE5 has previously been shown toaid in the quantification of T-cell subsets. To demonstrate the superior ability of the ZE5 Cell Analyzer to examine a diverse range ofbiological applications, exosome and immunophenotyping studies were performed. The exosomes were isolated and used to show thesubcellular detection capabilities of the ZE5. Published studies show measurements of cell health using more than 20 parameters at one
time with the ZE5; here we used peripheral blood to demonstrate simultaneous detection of 19 parameters. The ZE5 Cell Analyzer,
designed with an understanding of sample integrity and with a combination of speed, automation, and innovative design, will show that acytometer can be used for complex data analysis to easily resolve rare or dim populations with high-quality data, as well as routine
laboratory work.
How Does the ZE5 Cell Analyzer Help You Do More?
Unmatched Speed and Stability
The ZE5 Cell Analyzer combines improved speed, enhanced electronics, and a cell laser transit time that is five times
faster than other systems to create a unique and indispensable cell analysis system for any laboratory (Figure 1A).
The ZE5 Cell Analyzer can run at high speeds without compromising data quality due to enhanced fluidics that delivera stable flow rate up to 3.5 µl/sec. To match this rapid flow rate, the ZE5 Cell Analyzer’s faster laser transit timecreates a shorter pulse width, which is then sampled two times more frequently than comparable systems. The
increased sampling rate, coupled with fast, low-noise electronics, results in a tighter coefficient of variation (CV) andhigher-resolution data (Figure 1B). The speed of the ZE5 Cell Analyzer enables more in-depth investigation and more
specific analysis in applications, such as identification of specific cell subsets undergoing apoptosis, real-time
activation and proliferation of cells due to immune or drug response, or detection of cells in different stages of thecell cycle.
Obtain Better Reproducibility with No Carryover
Conventional flow cytometry requires manual washing of the sample probe between samples to reduce sample-tosample carryover. The ZE5 Cell Analyzer automates this activity with its flying collar wash feature that automaticallywashes the sample probe between samples. Automation of crucial steps reduces carryover and potential
contamination, thus removing a primary source of data variation (Figure 2) and providing results you can trust.
Study Small Samples with Ease
The ZE5 Cell Analyzer has small particle detection capability (forward scatter [FSC] from the 405 nm laser), permitting
analysis of difficult-to-study samples such as exosomes, which are generally smaller than 250 nm (Figure 3).
Exosomes are extracellular vesicles (EVs) that have been implicated in intracellular communication and are a key
area of interest in disease research. Conventional exosome analysis using flow cytometry can require manual
hardware adjustments and advanced instrument calibration. The ZE5 Cell Analyzer now provides a user-friendly
platform with built-in capabilities that allows any lab to study these and other small particles. Using both surface andinternal markers, centrifugation, beads, or other staining methods, the ZE5 displays optimal sensitivity for small
particle identification.
Publish, Don’t Rerun Experiments
The low-noise electronics in the ZE5 Cell Analyzer also contribute to great resolution and consistent, reproducible data
even at the high speeds only the ZE5 can achieve (Figure 4). The ZE5 Cell Analyzer instills confidence in results andenables quicker experiment-to-publication time. With reduced carryover, faster speed of acquisition, and multiple
parameters, the ZE5 Cell Analyzer supports the robust exploration of novel assays while improving the quality andreproducibility of ongoing experiments.
Analyze Cell Populations You Couldn’t Study Before
With up to five lasers and 30 detectors, the ZE5 Cell Analyzer easily improves upon standard assays, supports newpanels, and is an excellent aid in difficult-to-study rare cell analysis. The option for multiple spatially separated lasers
facilitates the study of highly complex samples. Numerous detectors expand options and allow for larger panels,
enabling critical data acquisition of whole samples (Figure 5). No longer do samples need to be split, which increases
the sensitivity of measurement.

ResultsABFig. 1. Speed and signal stability. A, the ZE5 Cell Analyzer outperforms other systems at higher acquisition speeds because it continues toacquire data in the 100,000 events per second range, whereas other systems drop off around 20,000 events per second. Dragon Green Beads(Bangs Laboratories, Inc.) were used in a serial dilution to determine when the observed acquisition rate falls off the theoretical limit. ZE5 CellAnalyzer (—); cytometer 1 (—); cytometer 2 (—); cytometer 3 (—). B, beads were read at increasing events-per-second rates, and singlet
beads were gated using pulse geometry gating. The percentage CV of side scatter (SSC) and fluorescein isothyiocyanate (FITC) was plotted
over approximately 4,000–129,000 eps. The mean and standard deviation (SD) of CVs over this range were FITC-A (●): mean = 10.6, SD =0.063; SSC-A (▲), mean = 11.4, SD = 0.063.

BACFig. 3. Exosome analysis. A, yellow fluorescent beads ranging from 0.22 to 1.35 µm were used to set up the voltage of a ZE5 CellAnalyzer; B, the size of exosomes purified from MCF-7 cells was determined by Zetasizer ZSP (Malvern Panalytical Ltd) to be in therange of 185 to 300 nm. Exosomes were gated on FSC from the small particle detector off the 405 nm laser vs. SSC. C, dual staining
with CD63-FITC and CD81-APC was performed using exosome-coated SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories,Inc.), which had been incubated with unconjugated Anti-Human CD63 (Bio-Rad) prior to the fluorescent conjugation for 30 min at room
temperature and washed twice with PEB–0.1% Tween 20.

Fig. 4. Robust reproducibility. Whole blood was lysed and stained with CD45–Alexa Fluor 488/CD3-BV421/CD8-APC-Cy7.
Twenty thousand events were acquired per run to evaluate various population percentages over multiple event rates.

AB

Laser/Filter Marker
Fluorophore
700LP CD25
BUV737387/11 CD45RO
BUV395355
447/60 Live/DeadZombie UV
525/50 CD19
BUV496420/10CD3Brilliant Violet 421
460/22
CD4V450625/24 CD20 Brilliant Violet 605
405 525/50 CD27
V500
750LP CD16 Brilliant Violet 785
720/60 CD123 Brilliant Violet 711
670/30 CD14 Brilliant Violet 650
488 525/35
IgD
Alexa Fluor 488
670/30 CD127
PE-Cy5615/24 CCR7PE–Dazzle 594
561577/15 CD11cPE750LP CD33
PE-Cy7720/60
CD8Alexa Fluor 700
640 670/30 CD56
APC
775/50 HLA DR
APC-H7Fig. 5. Large panel analysis. Whole blood was lysed and stained and 20,000 events were acquired to evaluate various populations.
A, chart showing a 19-color staining panel; B, representative plots showing data analyzed with Everest Software for T regulatory (T reg)and natural killer (NK) cell populations. Initial gates were set using Zombie UV Live/Dead Dye to exclude dead cells.

Conclusions
Using the ZE5 Cell Analyzer we can detect particles as small as exosomes to cancer cells to produce biologically
relevant data in a high-throughput manner. Our results with the ZE5 Cell Analyzer demonstrated:Fig. 2. Carryover reduction. Lysed whole blood was run on the ZE5 Cell Analyzer in high-throughput mode. After each sample, the system
performed an automatic wash cycle of 0.25 sec outside and 1.75 sec inside the sample injection port. A clean tube of water was run
immediately after the wash to evaluate carryover. Row A1 shows the populations acquired with the corresponding well A2 carryover
demonstrated underneath. Representative plots are shown for five separate runs.

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