High Throughput Single Cell Analysis - (HTSCA) Facility
Dr Jessica Rowley
In recent years there has been a very large increase in research involving single cell analysis, driven by an increasing awareness that bulk assays hide important biological phenomena and by the availability of new high-resolution equipment and techniques. In bacteria, this concerns issues such as phenotypic heterogeneity of virulence gene expression, cell surface composition, persister formation, antimicrobial resistance, immune detection, signalling and responses.
The ability to sort live pathogenic bacteria is essential for the analysis of complex heterogeneous populations and in 2014 we created the CMBI Flow Cytometry Facility. This comprises a BD FACS Calibur analyser and a state-of-the-art high speed BD FACSAria III sorter enclosed in a Class 2 microbiological safety cabinet with aerosol management. The Facility has been of immense benefit to CMBI researchers. It has already been used to separate and compare E. coli and Salmonella sub-populations for transcriptomics and antibiotic tolerance studies, screen a mutant library of P. aeruginosa for strains affected in iron uptake, analyse the interaction of virulence factors and host targets in infected cells, analyse the response of host cells to the heterogeneity of bacterial growth, track inflammasome activation upon bacterial infection, select clones for CRISPR/Cas9-based somatic knockouts, and isolate infected cells from the C. rodentiummurine model. Several papers involving use of the facility have been published and more are in preparation. In addition, international collaborations have been established, for example to develop chromosome conformation capture analysis of Salmonella and host cell sub-populations. The Facility was managed by Dr. Sophie Helaine, who had taken on responsibility for the day-to-day management of the instrument, training of users, safety and maintenance. Facility use has increased considerably over the last two years with cell sorting being undertaken on a daily basis for the benefit of CMBI members. To meet our growing need in this area the MRC provided funding for the support of a Facility Manager to maintain, train and help CMBI users with experiments and data analysis. Dr Jessica Rowley was appointed in April 2018 to manage the new HTSCA facility.
The new facility also includes high-throughput imaging equipment. We obtained funding for the purchase of a high-throughput live-imaging instrument, a fully motorised inverted microscope and a CellASIC® ONIX2 microfluidic system. After demonstrations of instruments from several companies, the procurement of the high-throughput imaging and the microscope is currently at the tendering stage.
The high-throughput automated imager will allow image-based assays and screens on both bacterial and host cells. Bacterial mutants can be identified rapidly by multi-channel fluorescence imaging-based screens in up to 1,536-well plates. Similarly, rapid screens for genes involved in host responses can be identified by RNA interference or CRISPR-based loss-of-function. Sophisticated studies involving responses of live CL2 bacteria to antimicrobials and other molecules and conditions in continuous, time-lapse experiments will be possible on this instrument.
An inverted microscope for high-resolution imaging using advanced auto-focus, quick multi-channel z-stacks and environmental control will provide subcellular resolution of host pathogen interactions and allow studies on single bacteria within the ONIX2 microfluidics chamber. Thus, the inverted microscope will provide high-resolution time-lapse imaging, while the high-throughput automated imager gives us speed and reliably repeatable protocols for larger screens. Dr Jessica Rowley will be trained in operating the imaging equipment and will help train and help other users. The facility will therefore have a highly trained manager who will be responsible for day-to-day operations, user training and providing technical help, calendar and budgetary maintenance of the all HTSCA equipment, including the flow cytometers and imagers.