From understanding immune responses to screening new drug candidates, flow cytometry plays a critical role in modern biomedical research. By rapidly analyzing thousands of cells per second, researchers can gather precise, multiparametric data at the single-cell level. Recent innovations in automation and high-throughput screening have further expanded the technology’s potential, making it a powerful tool for drug discovery.

Richard Cuthbert is a product manager at Bio-Rad with extensive experience in biomedical research, including stem cell biology, rheumatology, and immunology. Drug Discovery News spoke with Richard to discuss the evolution of flow cytometry, its advantages over other screening methods, and how next-generation instruments like the ZE5 Cell Analyzer are streamlining drug development.

What is a flow cytometer and what are its primary applications in biomedical research?

The body consists of hundreds of distinct cell types, each performing specialized functions. To understand how the body works, we must first understand its cells — which requires identifying and quantifying them. Flow cytometry can be seen as an evolution of microscopy, which excels at visually classifying cells but falls short in quantitative analysis. Measuring the relative proportions of different cell types under a microscope is both time-consuming and labor-intensive, making flow cytometry a powerful alternative.

Flow cytometry overcomes this limitation by analyzing cells as they flow through an optical system, eliminating the need for manual inspection. Instead of being fixed to a slide, cells are rapidly assessed and electronically counted, with tens of thousands measured every second. This high-speed processing enables precise statistical analysis and supports significant multiplexing, allowing researchers to identify and quantify dozens of cell types simultaneously.

Flow cytometry has a wide range of research applications, but its most common use is distinguishing and quantifying different cell types — a process known as immunophenotyping. This is an essential tool for understanding biology at the cellular level. Beyond immunophenotyping, flow cytometry can be used for assays measuring binding, apoptosis, activation, and cytokine production, among others. Much like microscopy, it’s an incredibly versatile technique.

How can high-throughput flow cytometry benefit drug discovery and development?

It’s important to define what we mean at Bio-Rad by high-throughput flow cytometry. In the past “high throughput” has been taken to mean that an instrument can process 96-well plates, regardless of the fact that a single 96-well plate could take several hours to complete. The ZE5 Cell Analyzer can process 96-well plates in less than 15 minutes and at the same time analyze 100,000 cells per well. It can also process 384-well plates in less than one hour and is designed to be integrated into robotic workcells, so it’s really a step change in the scale of what can be achieved.  

The ZE5 Cell Analyzer allows researchers to perform just about any assay at the speed of a screening instrument. Although screening cytometers have been around for a while, they have always been limited in their performance and not well suited to some types of flow applications, particularly assays that require a lot of cells or more complex immunophenotyping.

What parameters can be measured in a cell analyzer to assess drug response at the single-cell level? 

One of the great things about flow cytometry is that all of the data is at the single-cell level. There are a huge number of ways to use it to assess drug response.

Flow cytometry can be used to assess the effectiveness of a drug that targets cancer cells by quantifying those cells in a treated sample. It can also be used to detect apoptosis in specific cell types by applying dyes that identify apoptotic cells. Additionally, cytokine release or the expression of activation markers can indicate cellular activation. Some drug candidates may trigger an immune response — commonly referred to as immunogenicity — which can be detected by measuring increases in T cell populations. These are just a few simple examples and represent only a small fraction of what’s possible with flow cytometry.

Phenotypic screening builds on these types of assays but applies them differently. Instead of testing a few drug candidates across many samples, it involves screening a large library of compounds to identify those that produce a desired cellular response — such as proliferation, apoptosis, or activation. This approach is particularly valuable in small molecule drug discovery.

How does flow cytometry compare to other drug screening techniques, such as high-content imaging or enzyme-linked immunosorbent assay (ELISA)?

The optimal approach for any study depends on several factors. ELISA can be an excellent choice for screening, particularly when speed is a priority. However, it is limited in the number of parameters it can measure. Additionally, surface-expressed proteins may fold differently when removed from their native context — such as when bound to plastic — potentially affecting assay outcomes.

Flow cytometry allows binding assays to be performed in a much more biologically relevant context, minimizing the risk of false positive and false negative results.

High-content imaging is a powerful technique for researchers needing spatial or localization data, particularly when working with tissues or adherent cells. However, it can struggle with resolution and segmentation — accurately defining cell boundaries — which may impact its reliability for single-cell analysis. It’s also less suited to non-adherent cells, making it less suitable for assays involving blood or immune cells, where flow cytometry is more appropriate. Moreover, high-content imaging lacks the level of multiplexing achievable with flow cytometry; for instance, the ZE5 Cell Analyzer can measure up to 27 fluorescent parameters simultaneously.

Are there any further innovations in flow cytometry technology that are improving drug discovery workflows?

We’ve touched on automation already, but it’s worth highlighting here. Automating experimental workflows offers significant advantages — not just in time savings, but also in improving reproducibility.

Traditionally, flow cytometry hasn’t been closely associated with automation. One key reason is that many systems require ongoing manual supervision to operate correctly, which limits their compatibility with automated processes. While some instruments are technically automation-compatible, they weren’t designed with automation in mind.

The ZE5 Cell Analyzer, however, was built for automation from the ground up. Many of the steps that typically require manual oversight are handled internally. For example, the integrated plate loader can agitate samples, switch between different formats, and control temperature — all without user intervention. The instrument also monitors fluidics levels and remaining runtime, features automatic fault detection and recovery to handle blockages, and even performs a self-cleaning routine on shutdown. Many of these features are simply not available on other systems.

Another important factor is the quality of the application programming interface (API). The ZE5 Cell Analyzer features a vendor-agnostic, highly flexible API that allows integrators and researchers to fully harness the system’s capabilities.

With these features, automated flow cytometry holds strong potential to not only accelerate drug discovery but also improve its accuracy, reduce costs, and support the faster development of effective treatments.