NIH funded researchers at The University of Tennessee Health Science Center currently have critical need for a high-speed fluorescence activated cell sorter (FACS) with two or four-way sort option, the ability to sort into tubes or microplates, the option for single-cell deposition, detectors for forward and side scatter and ten fluorescence channels, temperature control for collected samples, and protection from aerosol dispersion. This application is for funds to purchase a Becton-Dickinson FACSAria FACS that is ideally suited to meet those demands. There is currently only one cell sorter in operation on the UTHSC campus. The cell sorter currently in operation is 14 years old and because of its age, is frequently out-of-service. New replacement parts for many mechanical and electronic components are no longer available and can only be acquired by scavenging from other similarly aged machines that are removed from service. Although the existing cell sorter was modified for high-speed sorting, it does not meet the minimum requirements indicated above. The existing sorter allows only four fluorescence channels, cannot sort into microwell plates, and cannot perform single-cell deposition. Since its inception in the late sixties and seventies, flow cytometry has become a mainstay for almost all research that involves the analysis of cells and cell functions. Monoclonal antibodies to the plethora of cell membrane markers, differentiation markers or CD antigens, and probes for cytoplasmic organelles, such as endosomes, nuclei, and mitochondria have made flow cytometry an even greater research resource. The uses of cell sorting by the investigators in this application include sorting macrophages that have ingested bacteria, sorting leukocytes into lymphoid and monocytic subsets, sorting specific T cell subsets based upon class II tetramer binding, isolating brain cells based upon level of expression of GFP reporters, isolation of lung endothelial cells before and after trauma, using recombinant, soluble T cell receptors to isolate insect cells expressing recombinant MHC class ll-peptide, and isolation of distinct B cell subpopulations based upon level of transgenic surface Ig expression. ? ?
| Brooks, Danielle L; Seagroves, Tiffany N (2018) Fluorescence-Activated Cell Sorting of Murine Mammary Cancer Stem-Like Cell Subpopulations with HIF Activity. Methods Mol Biol 1742:247-263 |
