Flow cytometry remains one of the most widely used techniques for single-cell analysis. However, cell-based assays have become increasingly complex, with modern drug discovery programs often requiring that immunophenotyping be integrated with measurements of cell health and function to generate data with greater biological relevance. This has highlighted a major limitation of traditional flow cytometry—namely that while it provides highly accurate identification of specific cell types within a heterogeneous sample, any additional analysis such as cytokine quantification must be performed separately. To overcome this problem, many researchers are switching to advanced flow cytometry systems that enable multiple assay principles for both cells and beads to be combined in a single well.
Many drug discovery workflows rely on bead-based immunoassays that are read by flow cytometers to identify and progress potential drug candidates based on how they affect a specific cell type. In a typical setup, the cells of interest are first isolated from a heterogeneous sample before being transferred to a microtiter plate where fluorescently coded beads are used as a solid support matrix for assessing molecular interactions. A main drawback of this approach is that it can rapidly create a bottleneck due to its low throughput. The transfer process also introduces the risk of data integrity becoming compromised, as well as requiring relatively large sample volumes to provide sufficient material for analysis.
A further disadvantage of analyzing cells and beads separately is that multiple analytical instruments are used to collect the data. In the example just described, a conventional flow cytometer would be employed for immunophenotyping and would additionally be used alongside various plate readers to assess cell health and function. Merging and analyzing distinct datasets is notoriously prone to error, not least because multi-step operations frequently involve different sample-preparation techniques and require the efforts of several laboratory personnel. There is also the danger that samples could be mislabeled during partitioning for different assay formats.
The need to perform immunophenotyping separately from monitoring cell health and function inevitably impacts experimental flexibility. With traditional flow cytometers being largely designed for tube-based sampling, long processing times, and high associated reagent costs often restrict researchers to running fewer replicates or including less controls than might be preferred. Moreover, where sample material is in short supply, it can frequently be necessary to decide upon which readouts are the most essential, meaning that valuable information is overlooked.
Traditional flow cytometry involves numerous manual processing steps and requires that researchers have a high level of expertise to quickly set up experiments and streamline data acquisition. As well as introducing user bias that can impact experimental reproducibility, this level of operator intervention can make traceability more difficult, especially where instrument settings are recorded in individual lab notebooks or where time constraints prevent personnel from noting key QC measures for each run.
Advanced flow cytometry systems overcome the limitations of traditional flow cytometry-based workflows by combining multiple assay principles for both cells and beads in an individual well. Now, researchers can analyze immunophenotype, cell health, and cytokine secretion in parallel, using sample volumes as low as 10 μL in 96 and 384 well plates with no requirement for dead volume. Assay miniaturization not only reduces sample and reagent requirements but also provides opportunities to analyze samples at different concentrations, obtain triplicate measurements, include more controls, or generate broader EC50/IC50 curves for more reliable data.
Because advanced flow cytometry systems are designed to be more intuitive, they improve experimental reproducibility by eliminating many common sources of variability. For example, in-built dynamic ranges remove the need to perform photomultiplier adjustments, while being able to store experiments as templates for future use maintains consistency around parameters such as gates, color compensation, and data metrics. Reproducibility can further be enhanced by connecting advanced flow cytometry systems to automation; this also increases throughput and safeguards data quality by tracking QC measures over time.
Another important benefit of advanced flow cytometry is that there is no need to export data for analysis. Instead, researchers can use integrated software for near real-time reporting and can readily visualize data to identify trends—for example, using heat maps to study a single metric across multiple plates or to quickly focus in on wells exhibiting user-defined characteristics. In combination, these capabilities of advanced flow cytometry promise to reveal novel insights more efficiently while preserving sample material that is often irreplaceable.
The iQue® 3 is an advanced flow cytometry system that combines cell immunophenotyping, cell health, and secreted protein (cytokine) analysis in every well of a microtiter plate. To learn more, visit sartorius.com/iQue3