Automating Flow Cytometry for Cell-Based Screening in Drug Discovery
Flow cytometry is a powerful tool for cell quantitation and analysis across a wide range of clinical and research applications.
Most commonly used in the fields of immunology and haematology, recent improvements in instrument automation and throughput mean that the technique is now emerging as an increasingly attractive high-throughput screening (HTS) platform in drug discovery too.
Flow cytometry is a widely-used analytical method that uses light to count and characterise heterogeneous cell populations. Highly valued for its ability to measure multiple parameters simultaneously, the technique enables vast amounts of data on large, phenotypically diverse cell populations to be rapidly collected.
Coupled with the fact that it is a non-destructive method that can be performed on live cells allowing further processing and analysis, it has become well-established as an important tool for both investigatory and routine analysis across a wide range of applications.
Its ability to quickly generate a wide range of data on cell properties means flow cytometry has been widely used for a number of applications in drug discovery, from target identification and validation, through to toxicological assessment (1). As a screening tool, its ability to measure cell properties on a single-cell basis makes it highly useful when analysing highly heterogeneous cell populations. This powerful functionality is in contrast to the analysis of cell lysates on plate readers, for example, where phenotypic differences are averaged out and essentially lost during measurement.
However, due to technical challenges around throughput and sample handling, its use has traditionally been limited to relatively low volume applications such as small target-based assays. As a result, the screening of even small compound libraries has traditionally proven time-consuming and practically challenging.
Recent advances in instrument design, automation and handling capacity are now enabling the wider use of flow cytometry for HTS applications. The commercial availability of instruments capable of handling micro-384 well plates and screening tens of thousands of cells per second are opening up a wide range of opportunities for HTS applications in drug discovery.
Coupled with novel automated sample handling systems, these developments are simplifying workflows, enhancing throughput and reducing cost associated with performing HTS assays. Indeed, the growing importance of flow cytometry in drug discovery is highlighted by the fact the technology will be a key focus of this year’s SLAS tradeshow (2).
Flow cytometry basics
Flow cytometry works by analysing the way individual cells interact with light. In a typical experiment, cells suspended in solution are sprayed into the instrument via a nozzle. This thin stream of cells is passed in front of one or more focused laser beams, with the resulting fluorescence emission and light scattering measured by detectors. As large numbers of cells can be studied in a short period of time, flow cytometry is capable of generating large volumes of statistically significant information very quickly (3).
Overcoming high-throughput screening challenges
Those choosing to employ flow cytometry for HTS applications in drug discovery have traditionally encountered four key challenges when using conventional flow cytometry technologies: low throughput; difficulties around the handling of large sample sizes; the need for a significant amount of equipment expertise; and the requirement for multiple third party software for data analysis.
In recent years instrument manufacturers have sought to overcome these limitations and have developed a range of solutions that meet these challenges head on.
Take IntelliCyt’s iQue Screener PLUS, for example, a high-throughput suspension cell and bead screening platform which the manufacturer claims is the fastest on the market. Not only does it offer increased speed and the capacity for cell and bead multiplexing, the system also enables reagent cost savings through the use of smaller sample volumes and a comprehensive analysis and visualisation software platform. These are valuable features when it comes to meeting the demands of high throughput screening applications.
“We’re seeing a growing number of pharma and biopharma customers using the iQue Screener PLUS to perform high throughput, high content assays,” says Joseph Zock, Senior Director of Product Management at IntelliCyt.
“Our users are taking advantage of the ability to generate high content data in a physiologically-optimised way for critically important immune targets and functions across the entire early drug discovery process, from target identification through to secondary screening.” High throughput is important, of course, but with analytical reproducibility another key priority in drug discovery, many customers are looking towards the reduced variability offered by fully automated flow cytometry workflows.
One manufacturer putting a significant focus on automation is Miltenyi Biotec. Its recently launched MACSQuant X system is capable of automatically processing the 384-well plates used for HTS applications and is designed to deliver consistent results with uptake volumes as low as 5μL per sample.
With the option to incorporate the process into liquid handling systems, the instrument offers the potential for a fully-automated workflow that minimises operator variability and is suitable for commercial scale environments, expanding the range of drug discovery applications that are possible.
Miltenyi Biotec has also developed a fully-automated flow cytometric fluorescence resonance energy transfer (FRET) assay suitable for highthroughput investigation of protein-protein interactions (4). The technique has been used as a standardised method for monitoring drug effects. Using an annotated substance library, the automated set-up was able to analyse 4000 FRET samples within two days on a single flow cytometer.
Commercially-available flow cytometry platforms
There are currently a wide variety of flow cytometry systems on the market, available from a number of different manufacturers.
The Intellicyt iQue Screener PLUS platform is an integrated software and reagent system that enables rapid, high-content, multiplexed analysis of cells and beads in suspension. The platform comes in three different configurations, utilising either two or three lasers, which allows for up to 13-colour channel detection, ideal for functional and phenotypic applications that require more flexibility in experimental design.
By maximising the detection and resolution of traditional and innovative reagent dyes, tandem dyes and fluorescent proteins, the iQue Screener PLUS platform delivers high-performance, multicolour analysis, and is able to measure up to 35,000 cells per second. The system is designed for use with 96-, 384- and 1536-well plates, and users can work with as little as 1μL of sample.
Miltenyi Biotec’s MACSQuant range of flow cytometers combines performance and robustness with highest convenience. The range includes the MACSQuant Analyzer 10, MACSQuant VYB and MACSQuant X systems. Utilising three lasers (405, 488 and 638nm), the compact, robust 10- parameter MACSQuant X flow cytometer is designed to facilitate the running of 10-parameter experiments in just 15 minutes for a 96-well plate, and less than 60 minutes for a 384-well plate.
The system comes with an integrated multisample analyser that can analyse up to 15,000 cells per second – making it well-suited for high-throughput applications – and is designed to produce consistent results with less than 0.01% carryover between samples.
BD Biosciences offers a wide range of flow cytometers for research and clinical diagnostics applications. The BD Accuri C6 PLUS is designed with ease of use in mind allowing both new and experienced flow cytometer researchers to benefit from enhanced sensitivity and reliability in their experiments. The system comes with two in-built lasers, meaning up to four fluorophores can be used at any one time. For walkaway convenience, the BD Accuri CSampler Plus automation accessory automates sample handling for applications using 48- and 96-well plates and 24-tube racks and is designed to be used in combination with the Accuri C6 PLUS.
The BD LSRFortessa X-20 cell analyser, which can be fitted with up to five lasers enabling the detection up to 20 parameters simultaneously, is designed to deliver high-performance multicolour analysis with an extremely compact footprint (30” by 20”). With its fully automated sample handling functionality, the LSRFortessa X-20 analyser can measure a 96-well plate in less than 15 minutes, with less than 0.5% sample carryover. Similarly, its novel FACSymphony cell analyser is designed to leverage the inherent benefits of flow cytometry while enabling the simultaneous measurement of up to 50 different cell characteristics using up to 10 different lasers, thanks to an innovative detection-array technology.
Merck offers a variety of flow cytometer technologies, from compact instruments for basic analyses, to more advanced equipment combining flow cytometry with microscopy. It’s Muse Cell Analyzer is a compact benchtop-sized instrument suitable for automated cell analysis, capable of analysing three parameters. The Guava easyCyte instrument is a microcapillary-based machine that allows detection of up to 12 parameters, and is capable of handling 96-well plates as well as sample tubes. Merck’s Amnis range of imaging flow cytometers combines the speed and sample size of flow cytometry with the resolution and sensitivity of microscopy in a single platform. The FlowSight and ImageStreamX are capable of generating qualitative and quantitative imaging data, with both systems allowing up to 12 parameters to be analysed.
The ACEA Biosciences NovoCyte system is a high-performance, cost-effective benchtop flow cytometer designed for all levels of users and all types of laboratories. The instrument is designed for the detection of up to 17 parameters with enhanced sensitivity and resolution, and its fully customisable laser and optical configurations offer a high degree of flexibility while providing complex cell analysis.
The intuitive build also allows for automated instrument maintenance functions and advanced data analysis capabilities for greater usability. In addition, the NovoSampler Pro autosampler is capable of analysing samples at an acquisition rate of 35,000 cells per second, and is compatible with single tubes, multi-tube racks and 24-, 48- and 96-well plates, with sample volumes ranging from 10μL to 5mL.
Beckman Coulter also offers a range of flow cytometry systems for research and discovery, diagnostics and industrial applications. The Gallios system, for example, comes with two to four lasers and can measure eight to 12 parameters. Additionally, its CytoFLEX platform is available in three models – the most advanced possessing up to six lasers, allowing for detection of up to 23 parameters. The system can also be expanded with a 96-well plate loader, facilitating higher throughput.
The future of the field
With simplified, high-speed workflows delivering improved reproducibility and reduced cost per screen, the increased adoption of automated, highthroughput flow cytometric applications looks set to continue.
“Over the next few years I believe we will see continued growth of fully-automated flow cytometry solutions, minimising operator influence and thereby minimising assay variations,” says Dr Martin Büscher, Head of Biophysics at Miltenyi Biotec. “At Miltenyi Biotec, we’re working hard to come up with novel automated solutions that will open up new options in high-throughput flow cytometry and will advance drug discovery applications across the field.”
Büscher believes that one of the biggest challenges will be convincing users to trust fully-automated solutions to do the job they have been doing manually for years. Ultimately, the time savings that high-throughput automation will bring and continued improvements in the reproducibility and reliability of flow cytometry data will play a key role in accelerating this transition.
Flow cytometry is an invaluable technique for the analysis of cell populations, and recent advances in instrument throughput and automation are enabling its wider use for a range of high-throughput drug discovery applications. The latest commercial machines are capable of measuring large numbers of parameters at impressive rates of tens of thousands of cells per second. Automated sample handling systems are simplifying workflows and making it possible to screen tens of thousands of compounds, and more, in a robust and resource efficient manner. DDW
This article originally featured in the DDW Winter 2017/18 Issue
Paul Avery is Managing Director and Richard Massey is a Science Writer at BioStrata, a life science specialist marketing agency. Its growing team in Cambridge (UK) and Boston (US) includes a significant number of people with deep scientific experience and knowledge. The agency offers everything from strategy, branding and message development through to creative, technical, digital, social media and PR execution.
1 Jepras, R and Ludbrook, S. Evolution of flow cytometry as a drug screening platform, Drug Discovery World, Spring 2013.
2 SLAS 2018 [Internet]. [cited 2017 Nov 28]. Available from: https://www.slas2018.org/.
3 Saeys, Y, Van Gassen, S and Lambrecht, BN. Computational flow cytometry: helping to make sense of high dimensional immunology data, Nature Reviews Immunology, 2016, 16, 449-462.
4 Kolontaj, K et al. Automated nanoscale flow cytometry for assessing protein-protein interactions, Cytometry A, 2016, 89, 835-843.