Flow cytometrical cell sorter

• Subject Area: Microbiology, Virology and Immunology
• Term: Funded in 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 450391471

The research groups involved in the present application are working in the area of basic immunology, infectious diseases, and tumor research and have a pivotal interest in understanding the complex interplay between different immune cell types. For this reason there is an urgent need to isolate the respective cell populations in high purity and quality from different tissues such as peripheral blood, secondary lymph nodes, liver, gut and other organ sites. As outlined in the respective subprojects, effective enrichment of these immune cell populations requires the combined analysis of a high number of extra- and intracellular markers. A cell sorter of the latest generation will enable many novel research projects that are currently hindered by the restricted number of sorting parameters available. Among the novel projects are the isolation of lymphocyte populations such as the recently described innate lymphoid cells (ILC) as well as subpopulations of natural killer, T, and B cells that are all characterized by complex surface receptor repertoires. Many complex staining protocols that are presently only applicable to flow cytometrical analysis on the respective multi color instruments could be directly transferred to the new cell sorter without the need for downgrading. The largely increased number of parameters measurable on the new instrument will greatly improve sensitivity of measurement by decreasing the cross talk between different channels due to minimized spectral overlap of the many newly available fluorophores. This is of special importance when analyzing transfectants expressing multiple fluorophore-coupled intracellular proteins, which requires high sensitivity and minimal spectral overlap for efficient sorting. The desired instrument is integrated in a class 2 safety cabinet, which is essential for several projects involving bacteria, intracellular parasites, and viruses. An additional novel feature of the new cell sorter is single cell sorting in 96- and 384-well format with retrospective analysis of all sorting parameters called index sorting, i.e. for every sorted cell important information such as granularity and fluorescence intensity is available and can be compared with subsequent cloning or sequencing results. Another highly interesting feature of the new instrument is chromosome sorting for subsequent long read sequencing and cost efficient de novo assembly approaches. The resolution of complex genomic regions encoding immunologically important gene clusters such as the MHC locus and the LRC/KIR locus on human chromosomes 6 and 19, respectively is dramatically improved by this enrichment step while greatly reducing the costs of studies on the population level as proposed by the applicants. Finally, the new cell sorter enables simultaneous collection of six different cell populations (6-way sorting) compared to four presently, which is especially important if sample material, for example from clinical specimens, is limited.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: 6-Laser Zellsortierer
• Instrumentation Group: 3500 Zellzähl- und Klassiergeräte (außer Blutanalyse), Koloniezähler
• Applicant Institution: Heinrich-Heine-Universität Düsseldorf
• Leader: Professor Dr. Markus G. Uhrberg