Enabling High Dimensional Single Cell Analysis Through 6-Laser Spectral Flow Cytometric Analysis

This application is submitted by the MRC Weatherall Institute of Molecular Medicine Haematology Unit. The equipment The Sony ID7000 (ID 7000) is a transformative, state-of-the art, full spectrum flow cytometer. It has been validated to measure 52 parameters (with the capability of detecting many more). Flow cytometry, a cornerstone technique for 50 years, has enabled the identification of new cell types and their function. It measures proteins and chemical characteristics of individual cells or particles, in complex mixtures. It does this by measuring light scatter and fluorescence from fluorochrome tagged antibodies and fluorescent reagents bound to the cell surface and intra-cellular components. The challenge being addressed The most advanced standard flow cytometers only accurately measure up to 30 different parameters. This limits their ability to uniquely identify multiple cell populations, especially rare populations, and their function, in complex cell mixtures, and how these populations respond to environmental changes. Furthermore, cell population diversity and heterogeneity of response to the environment changes with age and disease. Thus, being limited to measuring 30 parameters accurately, cheaply and quickly to identify cells with different function, especially rare cell populations, limits discovery science and is a challenge to overcome. The ID7000 addresses this challenge by measuring at least 52 parameters on single cells, assaying upto 2.4 million cells/minute. Context ID7000 will be located in the Flow Cytometry Facility, MRC Weatherall Institute of Molecular Medicine (WIMM), the largest such facility in the University of Oxford supporting the broadest range of advanced FACS cell analysers and cell sorters. The facility serves WIMM's two MRC Units (Molecular Haematology Unit, MHU and Translational Immune Discovery Unit, TIDU), Oxford's NIHR BRC and BTRU Cell and Gene Therapy Units, as well the whole University and local biomedical SME companies needing access to expensive infrastructure. The Facility, staffed by a manager with 30 years of experience and two flow cytometry specialists, provides expert support and training. Aim To enable a broad range of world-leading foundational basic science and translational discovery research in immunology and inflammation, haematology, DNA repair, cancer research, gene and cell therapy, stem cell biology and vaccines, relevant to human aging and improving human health. These programs are in the MRC Focus Themes of Precision Medicine and Advanced Therapies and UKRI-MRC strategic initiatives in Molecular and Cellular Medicine. Benefits 1) Study of at least 52 parameters for cell, and cell function, discovery and identification of cell phenotypes, especially rare phenotypes, through improved hardware (more fluorescent detectors), transformative software (novel spectral unmixing algorithms instead of spectral overlap compensation) that enable concurrent use of fluorescent antibody reagent combinations not possible on current conventional flow cytometers. 2) Transformed data quality as signals from individual fluorochromes are more clearly separated. 3) Detection of weak signals on highly auto-fluorescent cell populations. 4) Fast analysis of large cell samples (2.4 million cells/minute) with at least 52 parameters. A different method called mass cytometry can also measure at least 50 parameters but can only measure 0.2 million cells/minute. 5) 5-10-fold cheaper in overall costs compared to mass cytometry. 6) It will align with Facility's full-spectrum cell FACS sorter. Applications include: 1. Discovery of new cell populations, especially rare cell populations. 2. Discovery of new mechanisms of how cells respond to their environment. 3. Development if new methods of phenotypic and functional analysis in large cell populations.