This article and associated images are based on a poster originally authored by R. Mendoza, M. Santos, E. Dreskin, V. Kortisova-Descamps and R. J. Cuthbert and presented at ELRIG Drug Discovery 2025 in affiliation with Bio-Rad Laboratories, Inc.
This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.
Introduction
The ZE5 Cell Analyzer is a versatile, high-parameter flow cytometer. It has been featured in publications covering topics including antibody development (Momont et al., 2023), cancer (Ng et al., 2023), stem cell therapy (Crees et al., 2023), and molecular biology (DelRosso et al., 2023).
However, from these publications alone, it is difficult to appreciate how the unique characteristics of the ZE5 Cell Analyzer may have contributed to the quality and quantity of the data collected. Here, we examine three key capabilities of the ZE5 Cell Analyzer that make it the ideal choice for projects requiring large volumes of high-quality data.
We demonstrate how the ZE5 Cell Analyzer can be used to deliver data at a rate that is difficult to achieve with any other flow cytometer, without compromising data quality, in both bead- and cell-based assays.
Next, we demonstrate how fast sample transition can be combined with high event numbers across a 96-well plate, achieving minimal carryover without compromising assay speed. We illustrate how both these concepts can be applied to a functional assay involving monitoring of cell subpopulations following incubation with a CD20-specific monoclonal antibody.
Materials and methods
Flow cytometry
Unless otherwise stated, all antibody staining was preceded by incubation with Seroblock (Bio-Rad Laboratories Inc., #BUF070B) for 10 minutes at room temperature. Antibody staining was performed through incubation for one hour at room temperature in FACS buffer (PBS, Gibco, #14190144; 2 mM EDTA, Invitrogen, #15575020; 1 % BSA, Sigma-Aldrich, #A7284).
Following incubation, the cells were washed three times and then resuspended in FACS buffer. All wash steps were performed by centrifugation at 800 × g. For multicolor experiments, a single-stain control for each dye was included for spectral compensation.
All data collection was performed using the ZE5 Cell Analyzer (Bio-Rad, #12004279) and analyzed using FCS Express version 7 (DeNovo Software by Dotmatics).
Bead assays
A serial dilution of Dragon Green Beads (Bangs Laboratories, Inc.) was prepared. The observed acquisition rate, measured by the instrument, was compared to the expected acquisition rate, calculated from the concentration of beads in the sample and the sample flow rate.
High event rate analysis of PBMCs
Human peripheral blood mononuclear cells (PBMCs) were stained with Vivafix 649/660 Cell Viability Assay (Bio-Rad, #1351118) according to the manufacturer’s instructions. Cells were then incubated with antibodies: CD3 SBV515 (Bio-Rad, #MCA6146SBV515), CD4 SBV440 (Bio-Rad, #MCA1267SBV440), CD8 SBB615 (Bio-Rad, #MCA1226SBB615).
For data collection, cells were resuspended at 5 × 107 cells/ml. Three doubling serial dilutions were prepared, giving cell concentrations of 5 × 107, 2.5 × 107, 1.25 × 107, and 6.25 × 106 cells/ml and analyzed at a rate of 1 μL/second (n=5).
For data analysis, lymphocytes were identified based on forward and side scatter profiles, doublet exclusion was performed using side scatter pulse area and height analysis, and dead cells were excluded based on Vivafix 649/660 fluorescence.
Combining a high event rate with fast sample transition
Suspensions of stained and unstained Ramos cells (ATCC, #CRL-1596) were loaded onto a 96-well plate at a concentration of 14 × 106 cells/ml. Alternate columns of a 96-well plate were loaded with Ramos cells. Odd-numbered columns were loaded with 100 % unlabeled cells, even-numbered columns were loaded with 50 % unlabeled cells plus 50 % cells labeled with CytoTrack Green (Bio-Rad, #1351203).
Analysis was performed starting at column 1, row A, and moving horizontally to column 12, before moving down one row and restarting at column 1. This pattern was repeated until the position H12 was reached. All events over the threshold were considered to be cells.
For the calculation of percentage carryover, the number of positive cells observed in odd-numbered columns was compared to the total number of cells measured. This value was then doubled to account for the fact that 50 % of cells in even columns were positively stained.
High-speed analysis of a functional assay
Frozen PBMCs were defrosted, washed twice in complete RPMI 1640 (ATCC, #30-2001) with 1 % penicillin streptomycin (Gibco) and 10 % FBS (Cytiva, #SH30071.03HI) media, then resuspended in complete RPMI 1640 media at a concentration of 2 × 106 and incubated at 37 °C (5 % CO2) overnight.
The following day, cells were passed through a 40 μm filter and washed twice in complete RPMI 1640 media. Cells were then plated into a 96-well plate at 750,000 cells per well in a volume of 100 μL complete RPMI 1640 media with Rituximab biosimilar Anti-Human CD20 (Bio-Rad, #MCA6091), at concentrations of 100 μg/ml, 25 μg/ml, 6.25 μg/ml, 1.56 μg/ml, 390.06 ng/ml, 97.66 ng/ml, 24.41 ng/ml, and 6.10 ng/ml (n=5).
Cells were returned to incubation at 37 °C (5 % CO2) for four hours, then washed twice in PBS. Cells were stained as previously described using Vivafix 649/660 Cell Viability Assay, followed by incubation with primary antibodies (see Table 1). For data collection, cells were resuspended in FACS buffer at a concentration of 1 × 107 cells/ml, 10 μL of each well was collected at a sample rate of 2 μL/second.
Table 1. Reagents used. Antibodies and the live/dead dye are used for high-speed analysis of a functional assay. Source: ELRIG (UK) Ltd.
Axxx, Alexa Fluor; SBB, StarBright™ Blue; SBV, StarBright Violet; SBY, StarBright Yellow.
High event rate and data resolution
Fig 1. Acquisition rate demonstration on single samples. Serial dilutions of Dragon Green Beads (Bangs Laboratories, Inc.) were used to assess the observed versus expected event rate of the ZE5 Cell Analyzer (blue line) compared to three competitor instruments (grey, yellow, and red lines) (A). The coefficient of variance (CV) of the fluorescence intensity of a singlet bead population measured between 512.5–537.5 nm (yellow line) and side scatter (blue line) area parameters, at an event rate of up to 129,000 events/second (B). Acquisition time needed to acquire 40 million events at varying event rates, equivalent to detecting 400 rare events at a frequency of 1:100,000 (C). Image Credit: Image courtesy of R. Mendoza et al., in partnership with ELRIG (UK) Ltd.
High event rate and date resolution
Fig 2. Data conformity at varying event rates. Following the exclusion of debris, dead cells, and doublet events, the expression of CD3 was used to identify T cells, and subsequent CD4 and CD8 expression was used to distinguish T helper and cytotoxic T cells, respectively. Data were acquired at 6,250 events/second (A), 12,500 events/second (B), 25,000 events/second (C), and 50,000 events/second (D). The box and whisker plot shows the proportion of total T cells cumulatively as a percentage of lymphocytes and CD4-positive T helper cells and CD8-positive cytotoxic T cells as a proportion of total T cells, n=5 (E). Image Credit: Image courtesy of R. Mendoza et al., in partnership with ELRIG (UK) Ltd.
High event rate with fast plate handling
Fig 3. Data acquisition at a high event rate over a full 96-well plate. The flow cytometry gating strategy to identify total cellularity (A), remove doublet events (B), and identify CytoTrack Green-positive cells (C). Mean total cellularity in all wells (grey box), CytoTrack Green-positive cells (blue boxes), and negative cells (green boxes), in odd-numbered (100 % stained cells) or even-numbered (50 %+, 50 %– stained cells) wells (D). Carryover of CytoTrack Green-positive cells to odd-numbered wells containing only unstained cells (E). Image Credit: Image courtesy of R. Mendoza et al., in partnership with ELRIG (UK) Ltd.
High-speed functional assays
Fig 4. High-speed functional assay. Immunophenotyping gating strategy, B cells and T cells were identified based on the expression of CD19 and CD3, respectively. T cells were further subdivided based on the expression of CD4 and CD8, and CD56 was used to identify NK cells from the CD3, CD19 double-negative population (A). The number of B cells expressed as a proportion of viable cells incubated with varying concentrations of CD20 monoclonal antibody compared to isotype control, n=5, * denotes p<0.05 as calculated by two-way ANOVA test (B). All measured cell populations expressed as a proportion of viable cells incubated with varying concentrations of CD20 monoclonal antibody compared to isotype control, points indicate mean value, n=5 (C). Image Credit: Image courtesy of R. Mendoza et al., in partnership with ELRIG (UK) Ltd.
Conclusions
- High event rate analysis of up to 100,000 events/sec can be achieved without data loss or loss of data resolution
- Event rates of up to 50,000 events/sec are compatible with cellular assays without loss of resolution or instrument performance
- A 96-well plate can be processed in less than 15 min, whilst sampling more than 100,000 cells on average from each well
- High event rates and fast sample transition can be combined effectively to analyze complex functional assays
- These attributes are achieved without any deleterious effects, resulting in a synergism that allows virtually any assay to be performed at the speed of a screening instrument
- These insights offer potential benefits for laboratories struggling to meet user demand and open opportunities to expand the scope of investigations by freeing researchers from lengthy periods of data collection
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Last Updated: Dec 12, 2025
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