High-throughput flow cytometry and multiplex

Flow cytometry is a high-content assay technology providing information-rich multiparametric analysis on up to thousands of single cells per second. However, until recently, flow cytometry was only amenable to small-scale experiment because of slow sampling technology.

The development of fast, automated sampling technologies for use with flow cytometers has significantly increased the speed of this approach, rendering flow cytometry an attractive platform for screening and large combinatorial libraries of compounds.1 An ideal high-throughput flow cytometry system (HTFC) would be user-friendly with analysis software for multiparametric measurements and high-speed quantitative analysis of single cells and other particles.

How does automated sampling work in high throughput flow cytometry?

In HTFC, using an automated sample loading system multiple samples are aspirated from multiwell plates and delivered directly into the flow cell of a flow cytometer.2 The method involves the sequential extraction of samples from the wells into a single length of transfer tubing, where samples are separated from one from another by air gaps. The stream of samples from the entire plate is delivered directly into the flow cell of the cytometer. Moreover, microfluidic analysis systems and microliter sample volumes are of particular value due to the cost and often limited availability of test compounds.

Compared to other classical flow cytometry technologies with simple readouts, HTFC enables high content screening of multiparametric measurements in the more complex physiological environment of a cell. 

Multiplexing with flow cytometry 

Central to flow cytometry is the ability to perform multiple analyses on each cell or particle in a sample, known more commonly as multiplexing. The idea of multiplexing is fairly straightforward: different types of particles (eg, cells or beads) containing multiple fluorescent tags, are analyzed simultaneously in the same sample. One important advantage provided by multiplexing is the immediate gain in productivity, where, for example, the use of two or more fluorescent endpoints in one flow assay is equivalent to running two or more separate experiments on a plate reader. To this end, the gains in productivity and information per sample can be exponential.3

The last decade has seen several multiplexing strategies implemented:

  • Ligand-binding assay for screening novel binding events between multiple targets on individual cells, with each binding event detected by a different color fluorescent marker. 4
  • “Fluorescent cell barcoding,” which aims to uniquely label many populations of cells separately, combine them, and then deconvolve each population using analysis software. 5
  • Bead-based technologies, which can allow for hundreds-plex, assays in a bead-based ELISA format.6

Advantages of multiplexing

Multiplexing provides tangible gains in productivity by simultaneously measuring multiple assay endpoints. In addition to gains in productivity, multiplexing can generate detailed population analyses from samples containing mixed populations. Using cell type-specific fluorescent probes, it is possible to identify the subpopulations within a given sample and then query those subpopulations for further information.

To illustrate this point, an assay can be constructed that first identifies actively dividing cells and then measures the expression of a specific surface receptor protein within that subpopulation. In another example, lymphocyte subsets can be specifically labeled with fluorescent markers and then queried for expression of cell signaling proteins. In these examples, not only are there multiple endpoints of information within one sample but also differences within the sample can be exposed.


At Abcam, we have developed FirePlex-HT® for high-throughput, multiplex measurements of multiple analytes directly from biofluids. FirePlex-HT uses a two-step workflow and a no-wash, 384-well plate assay format, and is fast and automation-friendly with readout on high-content imagers, making it ideally suited for high-throughput screening studies.


  1. Kuckuck, F. W., Edwards, B. S. & Sklar, L. A. High throughput flow cytometry. Cytometry 44, 83–90 (2001).
  2. Ramirez, S., Aiken, C. T., Andrzejewski, B., Sklar, L. a & Edwards, B. S. High-throughput flow cytometry: validation in microvolume bioassays. Cytometry. A 53, 55–65 (2003).

  3. Black, C. B., Duensing, T. D., Trinkle, L. S. & Dunlay, R. T. Cell-based screening using high-throughput flow cytometry. Assay Drug Dev Technol 9, 13–20 (2011).

  4. Young S.M.Bologa C., Prossnitz E.R., Oprea T.I., Sklar L.A., Edwards B.S. High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands. J Biomol Screen 4, 374-82 (2005).

  5. Krutzik, P.O., Matthew R.C., Trejo A. & Nolan G.P. Fluorescent Cell Barcoding for Multplex Flow Cytometry. Curr Protoc Cytom 55, 6.31 (2011).

  6. Ding, M., Kaspersson, K., Murray, D. & Bardelle, C. High-throughput flow cytometry for drug discovery: principles, applications, and case studies. Drug Discov. Today 22, 1844–1850 (2017).

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