The NHGRI Flow Cytometry Core is open to all NHGRI and adjunct investigators. Currently, the Flow Core provides services to over fifty investigators from five of the six wet-lab branches within NHGRI. NHGRI investigators can meet with a member of the Flow Core for guidance about project development, sample preparation, execution of the experiment and other areas involving flow cytometry. We use an on-line calendar for scheduling time on the analyzers. Sorts are scheduled by e-mail or in person with Core personnel. The Core is staffed Monday through Friday for a wide variety of services including cell sorting, analysis of transfected cells, apoptosis assays, cell cycle analysis, viability assays, extracellular and intracellular antigen expression. Flow Core personnel perform all of the cell sorting and are often involved in cell analysis. The Flow Core trains investigators in the routine use of the analyzers and AutoMACS. Currently twenty Core trained users have access to the three analyzers. Through the use of a key card system, these investigators have access to the analyzers after routine hours and on weekends and holidays. The NHGRI Flow Cytometry Core was certified as a CLIA (Clinical Laboratory Improvement Amendments) lab in 2001 and currently operates under CLIA guidelines (most recent review 2010). CLIA tests offered include immunophenotyping, analysis of peripheral blood, apheresis products, and cell lines. Between January 1 and July 31, 2010 the Flow Core has performed over 350 sorts. These sorts have included the isolation of hematopoietic stem cells for investigators in GMBB who use these sorted cells for a variety of experiments including murine transplantation. Another frequent use of cell sorting is to isolate T-cells from immunodeficient patients and the study of T regulatory cells in both human and murine models for investigators in GMBB and GDRB. Investigators in GTB are sorting with the aim of obtaining complete transcriptional profiles of supporting cells in resting and regenerating neuromasts of zebrafish. They are currently characterizing a stable transgenic line, which is expressing GFP specifically in the neuromasts of the lateral line and in the nasal placodes. Other GTB investigators are sorting transfected cells derived patients with Hutchinson Guilford Progeria Syndrome. Many investigators use similar approaches to identify cultured cells expressing a marker gene after modification. Flow Core personnel perform approximately 15% of all analyses and assist or train others to do the remainder of the analyses. Between January 1 and July 31, 2010 the Flow Core has supported over 700 analytical experiments. Investigators from five NHGRI branches perform analysis of samples for cell cycle, apoptosis and BrdU incorporation. Investigators from GMBB and GDRB routinely use multi-color flow cytometry to analyze human and murine Lymphocyte and HSC samples. MGB is currently using the analyzers in their study of autophagic activity in a macrophage model of Gaucher disease. Cell lines over-expressing wild type or mutant glucocerebrosidase (the enzyme defective in Gaucher Disease) have stable expression of the GFP-conjugated autophagic marker LC3. A decrease in GFP signal is observed when autophagy is induced by nutrient starvation, which causes autophago-lysosome fusion. The acidic Ph of the lysosomes quenches the fluorescent signal, which is recorded by the analyzer. The High Throughput Screening module (HTS) on the LSRII can be used for running 96 well plates (vs tubes) or for high throughput screening. One application is the screening for secreted protein expression of Ly108 with the intention of identifying high protein secreting clones by intracellular staining in a 96 well format by GDRB. LSRII is also used for analysis of secreted protein by coating polystyrene beads with the supernatant of these clones (in a 96 well format) and screening these beads for antibody staining. Individual investigators receive training from the Flow Core in experimental design, sample preparation, instrument operation and data analysis. In the fall of 2009, the Flow Core added new technology to our existing instrumentation. We added a new laser scanning cytometer that allows analysis of a diversity of sample types (not all available by flow) including fixed cells, live cells, tissue samples (including sections or touch preparations) and tissue arrays. Some of the new assays include quantitative translocation assays, reporter assays, and DNA damage and repair analysis.
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