Cytometry User Group Meeting
Date: Monday, 29th January 2024
Time: 15:00 – 17:00, followed by a drink’s reception until 18:30
Venue: Sir William Dunn School of Pathology, MSTC lecture theatre, South Parks Road, Oxford OX1 3RE
Attendance is free, but please register in advance for catering and security purposes
Hybrid attendance is possible, invites will be sent out 24 hours prior to the meeting via the email provided at registration.
Cutting Edge Flow Cytometry for Pathogen Related Research
Javier Gilbert Jaramillo and Amy Napier
Nuffield Department of Clinical Neurosciences, and Sir William Dunn School of Pathology, University of Oxford
“Flow cytometry-based cellular modelling for the study of viral infection and pathophysiology.”
The use of hiPSC to study viral infections is of great attraction. However, hiPSC efficient differentiation and maturation still imposes complications in disease modelling. Zika virus (ZIKV), a neurotropic virus that primarily infects the fetal developmental brain, has been thoroughly studied since its outbreak in 2016. However, intrinsic cellular diversity within maturing neuronal cultures from hiPSC and the contribution of these cellular subtypes to ZIKV pathogenesis is yet to be determined. Similarly, studies on SARS-CoV-2 using hiPSC-derived models have failed to study cellular subpopulations within cultures that may display greater susceptibility to infection. Herein, we showed the advantage of using flow cytometry over currently used techniques such as immunocytochemistry for the characterisation of cellular subpopulations within cultures with potential relevance for viral infection and pathogenesis. Results showed that neuronal cultures comprising greater levels of undifferentiated neuronal progenitors were differentially susceptible to ZIKV infection, this mirroring epidemiological data. Moreover, flow cytometry-based analysis reflected a small subpopulation of macrophages with greater levels of SARS-CoV-2 entry receptor (ACE2) which may underlie disease-relevant mechanisms. Thus, the use of flow cytometry for the characterisation of cellular cultures may signify a powerful tool to study viral cell cycle and pathogenesis.
Engineered Biotechnology Research Group, Department of Engineering Science, University of Oxford
“Quantifying Bacterial Species in Co-Cultures using Natural Fluorescence.”
To realise the potential advantages of co-cultures over monocultures in bioproduction (e.g. broader substrate utilisation, increased yield/robustness), compositional control is needed to prevent fitness differences from causing one species to overwhelm another. Here, a two species E. coli – P. putida co-culture is dynamically controlled in a cybergenetic loop where a computer monitors the composition and adjusts their respective growth rates in real time. The relative abundance is inferred from OD measurements and fluorescence of a siderophore only produced by P. putida, while the growth rates are tuned by adjusting the temperature, a process that is automated on the Chi.Bio bioreactor platform. In order to build a model of the system and verify the population estimations from OD & fluorescence, flow cytometry was used to quantify the relative abundances of each species. While theoretically simple to distinguish, flow measurements required some optimisation due to the fact that the siderophore behaves differently to traditional fluorescent proteins, with certain conditions causing it to exit P. putida (creating false negatives) and even enter E. coli (creating double positives). In addition, experiments may be several days long with periodic sampling (e.g. every 2 hours), meaning samples had to be stored without significant signal deterioration
Carolyn Nielsen and Kirsty McHugh
Draper Lab, Vaccine Development and Antibody Immunology, Department of Biochemistry, University of Oxford
“B cell immunogenicity analyses and monoclonal antibody production using samples from malaria vaccine clinical trials.”
The Draper group in the Department of Biochemistry focuses on development of vaccines and monoclonal antibodies (mAbs) against blood-stage malaria parasites. Fortessa X20-based flow cytometry is used to interrogate human B cell responses following vaccination, while single cell sorting of antigen-specific B cells on the Aria Fusion enables monoclonal antibody production. Both approaches leverage fluorophore-conjugated vaccine antigen probes which bind to antigen-specific B cell receptors on the cell surface or, if cells are fixed, to intracellular antigen-specific antibody. This talk will highlight data from a recent trial testing two vaccine platforms for delivery of Plasmodium vivax malaria DBPRII: viral vectors (ChAd63, MVA) and protein/adjuvant (PvDBPII with Matrix-M). Using CITRUS and FlowJo, we report enhanced induction of DBPRII-specific plasma cell and memory B cell responses in protein/adjuvant versus viral vector vaccinees. Within protein/adjuvant groups, delayed boosting further improved B cell immunogenicity as compared to a monthly boosting regimen. This talk will also give an overview of ongoing mAb projects, including insights gained from a recent analysis of over 200 human IgG mAbs against the P falciparum vaccine candidate RH5.
Product Manager, Cell Analysis and Sample Preparation, Miltenyi Biotec
“Multiparameter flow analysis of antigen- and virus-specific T cells.”
It cannot be underestimated how important flow cytometry and flow sorting can be in gaining a better understanding of viral infection, in particular COVID-19. For example, analysing the immune cells might help us to understand why some patients show severe complications while others are nearly asymptomatic. MACS® Flow Cytometry Kits and REAfinity™ Recombinant Antibodies are optimized for lowest background, highest reproducibility, and most precise results. Our MACSQuant® Tyto® Cell Sorter allows gentle and sterile sorting of rare virus-specific T cells in a closed aerosol-free cartridge system.
Thanks to all those who attended our previous meetings. With special thanks to the speakers for making them such interesting and successful evenings.