The NHGRI Flow Cytometry Core currently maintains two BD FACSArias that are primarily used for sorting cells. Each FACSAria is configured with three lasers and can measure up to nine fluorescent parameters as well as physical parameters (size and granularity). Among the various applications over the past year, these sorting capabilities have been used for isolation of tissue cell populations for animal transplantation experiments;isolation of blood cell sub-populations for analysis of functional cellular properties and gene transcription profiles, as well as high throughput screening of enhancer regions. The FACSArias typically have a two week sign up waiting time and are currently used at capacity. For direct flow cytometry analysis applications not involving cell sorting, the Flow Core offers two BD FACSCaliburs and one BD LSRII. The FACSCaliburs can measure up to four fluorescent parameters, and are routinely used for data acquisition and pre-sort analysis. Due to budget restraints, FACSCalibur E0807 has been removed from service contract saving the Core $10,000 in FY11. Typical uses of the FACSCaliburs include analysis of GFP expression and cell cycle, as well as mutagenesis screening. The use of the FACSCaliburs has decreased as more investigators move to the BD LSRII for their analysis. It is anticipated that in the near future the Core will need to replace one of the FACSCaliburs whose technology is becoming obsolete with either a second LSRII or a FACSVerse. Both of the latter instruments are digital, multi-color analyzers. The BD LSRII uses digital electronics and Diva software similar to the Aria and is used to perform 9-color analyses. This allows investigators to characterize cells in more detail before sorting on the Aria. The instrument is equipped with a High Throughput Sample (HTS) device that is routinely used and gives the investigator the ability to analyze many samples in 96 well plate format without the need of sitting at the instrument during the acquisition procedure. The BD LSRII is available during regular Core hours as well as after hours and on weekends. Nonetheless, this instrument is currently used at maximum capacity, which may become an issue in the future. The Flow Core maintains a laser scanning cytometer (LCS). The LSC uses laser-based opto-electronics and automated analysis capabilities to simultaneously and rapidly measure biochemical constituents and evaluate cell morphologies. Current applications (80%) are focused on zebra fish projects including drug screens and blood development. The Flow Core also maintains a Miltenyi Auto MACS that is used for magnetic cell separations. The AutoMACS is often used as a pre-enrichment step prior to sorting on a flow cytometer. Over the past year, the services and capabilities of the NHGRI Flow Core have been taken advantage of by over 60 trainees from 5 Branches/14 Sections in the Institute. In addition, the Flow Core continued to maintain CLIA accreditation in support of immunophenotype and protein expression studies used in NHGRI clinical research protocols.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Scientific Cores Intramural Research (ZIC)
Project #
1ZICHG200350-04
Application #
8350172
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2011
Total Cost
$596,227
Indirect Cost
Name
National Human Genome Research Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Vilboux, Thierry; Malicdan, May Christine V; Chang, Yun Min et al. (2016) Cystic cerebellar dysplasia and biallelic LAMA1 mutations: a lamininopathy associated with tics, obsessive compulsive traits and myopia due to cell adhesion and migration defects. J Med Genet 53:318-29
Gomez-Rodriguez, Julio; Meylan, Fran├žoise; Handon, Robin et al. (2016) Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4. Nat Commun 7:10857
Psaila, Bethan; Barkas, Nikolaos; Iskander, Deena et al. (2016) Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways. Genome Biol 17:83
Pathak, Anand; Pemov, Alexander; McMaster, Mary L et al. (2015) Juvenile myelomonocytic leukemia due to a germline CBL Y371C mutation: 35-year follow-up of a large family. Hum Genet 134:775-87
Yokoyama, Tadafumi; Yoshizaki, Ayumi; Simon, Karen L et al. (2015) Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice. PLoS One 10:e0139729
Gomez-Rodriguez, Julio; Wohlfert, Elizabeth A; Handon, Robin et al. (2014) Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells. J Exp Med 211:529-43
Paralkar, Vikram R; Mishra, Tejaswini; Luan, Jing et al. (2014) Lineage and species-specific long noncoding RNAs during erythro-megakaryocytic development. Blood 123:1927-37
Rodriguez-Gil, Jorge L; Larson, Denise M; Wassif, Christopher A et al. (2013) A somatic cell defect is associated with the onset of neurological symptoms in a lysosomal storage disease. Mol Genet Metab 110:188-90
Zhao, Ling; Melenhorst, Jan J; Alemu, Lemlem et al. (2012) KIT with D816 mutations cooperates with CBFB-MYH11 for leukemogenesis in mice. Blood 119:1511-21
Hsu, Amy P; Dowdell, Kennichi C; Davis, Joie et al. (2012) Autoimmune lymphoproliferative syndrome due to FAS mutations outside the signal-transducing death domain: molecular mechanisms and clinical penetrance. Genet Med 14:81-9

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