Flow Virometry Sorting, B cell Cloning and Evaluation - CytoFLEX SRT and Viral Research

By John Tigges, Principal Associate in Medicine at Harvard Medical School,

James McCracken, Product Manager at Beckman Coulter Life Sciences, and

Cecilia Cavazzoni, PhD, Research Fellow at the Sage Lab, Transplantation Research Center at Brigham and Women's Hospital

Virus-like particles (VLPs) are nanostructures that possess diverse applications in therapeutics, immunization, and diagnostics. They consist of one or more different molecules, have the ability to self-assemble, and are virus particle mimics in that they share the same form and size. However, they lack the proper genetic material to be infectious.

Recently, VLPs have garnered increased attention in relation to SARS-CoV-2 vaccination. Due to their ability to elicit an immune response, VLPs have become an attractive alternative to conventional vaccination methods.

However, contaminants from host cell production are a major issue. Using a combination of flow virometry, as described by Tang et al., and nano-flow cytometry, one can isolate and characterize VLPs more efficiently using the CytoFLEX SRT benchtop cell sorter to selectively capture VLPs of interest. In addition, upon vaccination, it has become tantamount to measure immune response and the associated pathways.

Within the mechanism of SARS-CoV-2 vaccines, both follicular helper T (Tfh) and follicular regulatory T (Tfr) cells have an effect on germinal center (GC) reactions. GC reactions generate antibodies and memory B cells, and are regulated by a balance of Tfh and Tfr response.

In order to study this delicate response, single B cell clones from SARS-CoV-2 immunized mice were sorted into 96 well plates using a CytoFLEX SRT benchtop cell sorter prior to further downstream applications. Specificity, affinity, and somatic hypermutation were assessed via BCR sequencing and ELISA. 

Learning objectives:

  • VLPs: What are they and how are they beneficial to research?
  • The immunologic response to SARS-CoV-2 and associated pathways
  • Sorting as a tool for vaccine research

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Research Use Only, Not for use in diagnostic procedures.

About the speakers

John Tigges

John Tigges is the Director of the Flow Cytometry Science Center at Beth Israel Deaconess Medical Center and Principal Associate in Medicine at Harvard Medical School. He has been working in the field of flow cytometry for over 25 years and has extensive experience in cell sorting, multiparametric panel design, and small particle detection. His latest research in the detection of extracellular vesicles has allowed him to co-chair a nano-flow cytometry standardization group and become a member of the ISEV/ISAC/ISTH EV working group. He has worked with CytoU and ISAC Education Task force, to advance the flow cytometry knowledge base and is a member of the Instruments for Science task force which assists in bringing flow cytometry equipment and education to underserved communities.

James McCracken

James McCracken has over 20 years of experience in flow cytometry and is currently the Product Manager for cell sorters at Beckman Coulter Life Sciences. James has broad background in flow cytometry, initially a tool used in his graduate research then as a shared resourced facility manager, with specific expertise in cell sorting. He later joined the Beckman Coulter Team supporting customer training for sorters and analyzers. In his current role as the Product Manager, he takes great enjoyment from understanding what he can do to make more pleasant and intuitive instruments.

Cecilia Cavazzoni, PhD

Cecilia Cavazzoni is a PhD in Immunology and a Research Fellow at the Sage Lab in the Transplantation Research Center at Brigham and Women’s Hospital. She has been working on B cell biology and the regulation of adaptive and humoral immune responses to viral infections, vaccines and in the context of transplantation and autoimmunity.

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