The Core is actively involved in a variety of applications including the isolation of tissue and blood cell populations for animal transplantation experiments; isolation of cell sub-populations for analyses of functional cellular properties and gene transcription profiles, as well as high throughput screening of enhancer regions. Instrumentation: The Core is equipped with two Becton Dickinson FACSARIAs which can identify up to 13 fluorescent parameters and sort into tubes, 96 or 386 well plates, or slides; one Becton Dickinson LSRII which is used for direct high dimensional flow cytometry analysis applications (13 color), including high throughput analyses; and one four color BD FACSCalibur for more basic analyses. The Flow Core maintains a laser scanning cytometer (LCS). Current applications (100%) are focused on zebra fish projects including drug screens and blood development. An autoMACS Pro Separator is used for high-speed magnetic cell isolation of multiple samples. The autoMACS Pro Separator is designed for cell isolation in a fully automated, walk-away fashion. Current issues: We have upgraded both sorters to have 13-color capability and have introduced index sorting (Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways Bethan Psaila1,2,3, Nikolaos Barkas3, Deena Iskander1, Anindita Roy3,4, Stacie Anderson5, Neil Ashley3,Valentina S. Caputo1, Jens Lichtenberg2, Sandra Loaiza1, David M. Bodine2, Anastasios Karadimitris1,Adam J. Mead3* and Irene Roberts3,4*.) The index-sorting feature enables the investigator to sort cells into single wells of plates, and easily identify them on plots, in statistics views, and in a detailed results file. These features allow the rapid isolation and characterization of single cells for use in single cell RNA-seq, Fluidigm and other genomic analyses. The Core is approved as a BSL2 laboratory and is currently sorting of samples or samples from virally infected mice. Concerns include the high usage of the LSRII. This instrument is currently at maximum capacity and is used seven days/week including evenings, reflecting the high demand for this type of instrument and high dimensional single cell analyses. Due to the demands on the LSRII we feel that it is in the best interest of the Institute if we add an additional four-laser analyzer with an integrated plate reader to the Cores instrumentation. This analyzer would allow investigators who have limited access to an analyzer to increase their productivity and scientific output. Additionally, we would like to add a 561nm laser to the second sorter thereby increasing the utility of this instrument and bring it to current standards. The incorporation of the 561-nm laser into the BDFACSAria will provide the capability to better separate the excitation and emission of two commonly used fluorochromes, FITC and PE, resulting in the elimination of spillover between the emission spectra of these fluorochromes. In addition, the 561-nm laser efficiently excites the latest fluorescent proteins such as mCherry and dsRed, allowing the core to provide appropriate current technology to the members of the Institute. The Flow Cytometry Core is currently staffed by Stacie Anderson (Core Director, 100% effort, GMBB), and Martha Kirby (100% effort, GMBB). These investigators run the sorters, maintain the analyzers and other equipment and provide expert advice on design and execution of experiments.
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