Faster, better, and more of it – what’s next for flow cytometry–based screening?

This paid-for advertorial by Bio-Rad appeared in the DDW ebook – Innovating in the lab: an essential guide to drug discovery technology.

In the following article Richard J Cuthbert, PhD, Global Commercialisation Product Manager, Flow and Antibodies, Bio-Rad Laboratories examines the rise of flow cytometry as a screening tool.

In recent years, the use of flow cytometry in drug discovery has become widely accepted. It offers an unparalleled combination of speed, sensitivity, and multiparameter analysis, making it an essential tool for antibody screening and phenotypic drug discovery.  

The emergence of flow cytometry as the foremost technology for screening has been an incremental process. Early flow cytometers were large, difficult to operate and had comparatively few parameters. While significant improvements have been made, one fundamental limitation persisted; speed. 

Early innovations, like the addition of plate loaders or tube carousels, were an attempt at addressing the low-throughput rate associated with manual sample handling, but despite these, flow cytometry paled in comparison to other technologies like ELISA, where results could be rapidly collected from microtiter plates. The fundamental problem was that flow cytometers typically rely on each sample being drawn through a section of capillary tubing, before being focused into a stream of particles and presented one by one to a single set of detection optics. 

Figure 1: The ZE5 Cell Analyzer shown with a simple robotic workcell.

The breakthrough came with the idea that rather than drawing a single sample at a time, and then clearing the line, multiple samples could be drawn one after another into the sample line. In order to differentiate one sample from the next, an air bubble is introduced between the two samples. This approach has key advantages, as it facilitates a reduction in the sample volume and a corresponding increase in the concentration, thus increasing the number of samples that can be processed in a given time. It also allows the miniaturization of assays meaning a significant reduction in running costs. 

The power of flow cytometry 

With this innovation, the power of flow cytometry could finally be realized in a format that is amenable to high-throughput screening. Rather than being limited to a single parameter, as is the case with ELISA, researchers could apply multiple parameters to their assays and save huge amounts of time, by in effect performing two or more assays in the same well. Another great advantage is that assays can be performed using live cells, meaning targets, which would normally be expressed on the cell surface, are not subjected to any conformational change and binding epitopes are preserved. Compared to technologies such as imaging cytometry, what was once the Achilles’ heel of flow cytometry became one of its greatest strengths. The fact that particles are presented to the detection optics one by one means that data collection is at the single particle/cell level and not subject to resolution limitations. Because of this, flow cytometry does not rely on population averaging, meaning unwanted events can easily be discounted from analysis, unlike imaging cytometry.  

Today, some features are generally accepted as essential for a screening flow cytometer. These include compatibility with high-density microtiter plates, low sample volumes, suitability for automation, and above all, short acquisition times. However, just because an instrument delivers on these points does not mean that all challenges are met. Excessive sample carryover, downtime caused by instrument blockages, labour intensive file separation, and lack of flexibility can slow down projects. As can limitations on the number of excitation wavelengths and available detection channels. These are among the unresolved issues that limit the capability of some screening instruments. 

The future of flow cytometry–based screening 

The ZE5 Cell Analyzer from Bio-Rad has been designed to break through screening limits. It has a plate loader with built-in temperature regulation (4°C-37°C), orbital shaking calibrated to plate type, and is compatible with a wide range of sample input types. This makes it the most capable plate loader available today. As you would expect, it uses air gaps to separate samples in the sample line so that it is able to move between samples incredibly quickly. Unlike other systems, it combines this ability with industry leading detection electronics, making it capable of collecting up to 100,000 events/sec with no data loss. This is the difference between a system that can power through detection of rare events in a screening assay and one that simply cannot.  

Figure 2: Samples are drawn into the sample line and separated from each other by air and water gaps to facilitate rapid sampling.

When it comes to robotics, there is nothing holding you back. The ZE5 Cell Analyzer has an application programming interface (API) which is designed to be broadly compatible. It is vendor agnostic, LIMS friendly, and can be seamlessly integrated into new or existing workcells from a simple robot arm and plate hotel, up to the most complex multi-instrument platform. 

So the ZE5 Cell Analyzer delivers on the essentials and more than ever before. But what about those previously unmet challenges? If a system uses a probe that moves between samples, it’s a potential source of carryover. Recognizing this, the ZE5 Cell Analyzer includes a cleaning station that quickly washes the probe both inside and out, after each and every sample. This means that even at top speed, carryover is minimized to less than 0.5%.  

Blocking has always been an issue for flow cytometry and can lead to down time. One of the ZE5 Cell Analyzer’s stand-out characteristics is the quality of its fluidic system. This by itself significantly reduces the risk of blockage. Additionally, it has a reversible sample pump, meaning that even if a problem does develop, it can be addressed quickly and easily. 

When it comes to the time to collect your data, forget about spending hours separating individual samples from one big file. The ZE5 Cell Analyzer separates individual well FCS files on the fly, so that by the time your plates have completed their run, all your data will be ready to go. Unlike other systems that compromise data resolution, especially at low signal strength, data quality remains excellent, across the full dynamic range. 

In terms of flexibility, the ZE5 Cell Analyzer is second to none. With up to 27 fluorescence channels, you can handle much more complex assays and get more done in each single well. With five lasers, you can avoid problematic compensation, simplifying your experiments and preserving data quality.  

In summary, Bio-Rad’s ZE5 Cell Analyzer represents the next step in drug screening with faster data acquisition and data processing, better sample handling and data quality, more parameters, more lasers, and more and better results.  

Visit bio-rad.com/CellAnalysis to learn more about the ZE5 Cell Analyzer.  

About Bio-Rad

Bio-Rad has over 60 years of experience supporting life science research and clinical diagnostics. We are dedicated to offering tools and services to streamline assay development and manufacturing, to minimize downtime and provide the right answer the first time. Depend on Bio-Rad for tools, technologies, and expertise to enable cellular, genomic, and proteomic analysis. 

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