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When you hear the words “screening” or “high-throughput” in flow cytometry, what comes to mind? For many scientists, high-throughput flow cytometry screens are associated with drug or compound libraries, but did you know that you can also apply this technique to study microorganisms, clinical melanoma samples, and even protein trafficking? Read on for five examples of the use of flow cytometry in diverse, high-throughput screening applications.

1. Studying immunogenicity using flow cytometry

As most modern vaccines focus on the delivery of discrete antigens rather than entire organisms, vaccine development usually begins with identification and selection of antigens (Cunningham et al. 2016). This is the case whether developing oral poliovirus vaccines, injected ebola vaccines, or anything in between. During both antigen selection and preclinical and clinical testing, it is imperative to test for immunogenicity, safety, and efficacy. This requires an instrument capable of rapidly screening antigens. Specifics about this process are detailed by Dr. da Silva Antunes in his talk, “Development and Validation of a Bordetella pertussis Whole Genome Screening Strategy,” where he also details how a flow cytometer capable of quickly processing plates saves time.

Studying immunogenicity using flow cytometry

2. Characterizing microorganisms using a plate-based flow cytometer

Studying bacterial strains is no small feat, as demonstrated by a recent paper on the characterization of bacteriocinogenic strains (Fugaban et al. 2021). In a timecourse study of numerous strains grown at six different temperatures, authors needed to examine cell counts, growth kinetics, and fermentation. For this, they turned to a flow cytometer with plate capabilities to facilitate easy, high-throughput evaluation.
Characterizing microorganisms using a plate-based flow cytometer.

3. Screening protein trafficking processes efficiently with flow cytometry

Proteins play essential roles in “every signaling and regulatory process in biology” (preprint: Coukos et al. 2021). Who can argue with Robert Coukos and colleagues’ opening statement in their paper on protein trafficking in high-throughput screens? This paper discusses the development and use of HiLITR, a molecular tool that enables assessment of protein localization using a fluorescent readout. By combining HiLITR with flow cytometry and CRISPR interference (CRISPRi), the authors were able to perform a genetic screen for genes involved in protein trafficking. The authors also used flow cytometry to select for construct expression levels while generating clonal cell lines. The need to screen cell lines and constructs quickly and efficiently is the focus of flow cytometry with integrated automation.
Screening protein trafficking processes efficiently with flow cytometry

4. Monitoring immunotherapy patients using high-throughput flow cytometry

Personalized methods to treat patients with immune suppression have been shown to elicit substantial responses. An example is the treatment of cancer patients with immune checkpoint blockade antibodies, which can reinvigorate T cells. This immunotherapy works in some but not all patients, so early biomarker detection should be used to identify patients who would benefit the most. Flow cytometry can provide a solution, generating data on peripheral blood immune cells which can predict treatment outcomes after immunotherapy. The technique could also monitor a patient’s response during immunotherapy, helping to inform treatment decisions (Sanjabi and Lear 2021).

The tumor environment is constantly being explored and studied via flow cytometry. In a Bio-Rad app note from 2019, researchers focus on T-cell activation and exhaustion, a prospective target for cancer immunotherapy (Bio-Rad bulletin #7125). In this app note, the authors monitor T-cell exhaustion by examining surface markers, cytokines, and looking at long noncoding RNA (IncRNA) targets. Their findings included changes to expression of the checkpoint inhibitors PD-1 and TIM-3.

Monitoring immunotherapy patients using high-throughput flow cytometry

5. High-throughput compound screening at the single cell level

The use of flow cytometry for high-throughput compound screening is ever popular, including in COVID-19 research (Ng et al. 2020). As more labs begin using these techniques, protocols for compound screening remain in high demand. One recently published protocol uses flow cytometry to quantify cell surface expression of a target (Spangenberg et al. 2021). Realizing that analyzing protein expression at the single cell level can be demanding when you have large numbers of samples, the group describes a methodology for screening ~200,000 compounds, which they tested using THP-1 cells and compounds that regulate PD-L1 expression.

High-throughput compound screening at the single cell level
Whether performing immunogenicity studies, vaccine development, phenotypic drug discovery, therapeutic antibody screening, protein trafficking, or cancer biomarker discovery, science requires the processing of very high numbers of samples. These assays can benefit from high-throughput and high-parameter flow cytometry with integrated automation.

Meet the ZE5 Cell Analyzer and maximize your output. It’s not just another high-color flow cytometer — it’s the long-awaited solution for your high-productivity needs with the added benefits of superior performance and unparalleled flexibility. Try any of these high-throughput screening applications using the ZE5 Cell Analyzer today.


Coukos R et al. (2021). An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput screens. bioRxiv. Preprint., accessed August 23, 2021.

Cunningham AL et al. (2016). Vaccine development: From concept to early clinical testing. Vaccine 34, 6,655–6,664.

Fugaban JII et al. (2021). Characterization of partially purified bacteriocins produced by Enterococcus faecium strains isolated from soybean paste active against Listeria spp. and vancomycin-resistant enterococci. Microorganisms 9, 1,085.

Ng K et al (2020). Preexisting and de novo humoral immunity to SARS-CoV-2 in humans. Science 11, 1,339–1,343.

Sanjabi S and Lear S. (2021). New cytometry tools for immune monitoring during cancer immunotherapy. Cytometry B Clin Cytom 100, 10–18.

Spangenberg S et al. (2021). Protocol for high-throughput compound screening using flow cytometry in THP-1 cells. STAR Protocols 2, 100400.

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