Frederick CCR Flow Cytometry Core
Cancer and Inflammation Program (CIP)

Comprehensive support for studies of cell immunophenotyping, cell sorting, cell cycle, apoptosis and cell proliferation

Mission: Flow cytomety (FCM) is a unique experimental technology, which provides rapid, quantitative, multi-parametric, single cell analysis and separation. The mission of the flow cytometry core encompasses basic research support provided to members of the Frederick-CCR community. The laboratory staff also participates in the development of flow cytometry applications and resources for use in basic research. Resource development in the lab includes advances in instrumentation, methodology and computer/software. The core staff also trains investigators to operate the analyzer instruments. This laboratory is contract-operated with oversight by a user based steering committee.

Access: Dedicated to CCR and Program

Staff:
CCR Core Supervisor - Kathleen Noer, BS noerk@mail.nih.gov 301-846-5811
Support Staff - Roberta Matthai, BA rmatthai@mail.nih.gov 301-846-5811
                     - Guity Mohammadi, BS guitymoh@mail.nih.gov 301-846-5811

Expertise:
The CCR Flow Cytometry Core provides multi-color flow cytometry analysis of live or fixed single-cell suspensions derived from various tissues and cell cultures. The group can offer up to 6 parameter cell surface markers coupled with calcium-flux or cell cycle analysis. Standard and high-speed cell sorting of single-cell suspensions of up to 6 colors is available in either bulk sorts or by single cell deposition in 96-well plates. Cell cycle analysis software is available for cell ploidy, cell cycle, and apoptotic analysis of cell populations.

Established Technologies:

• Immunophenotyping of mouse and human cells using up to 12 parameters
• Apoptosis assays using annexin V, tunnel, caspase activation and mitochondrial membrane potential changes
• Cell cycle analysis and sorting
• Cell sorting using up to 8 colors
• Phenotyping of cells in proliferation assay
• Sorting and analysis of cells expressing fluorescent proteins

Instrumentation:

• BD FACSAria SORP cell sorter with four lasers (excitation lines at 488nm, 637nm, 405nm and 355nm), 12 fluorescent detectors, high speed four-way bulk sorting and single cell deposition sorting
• BD FACSAria cell sorter with three lasers (excitation lines at 488nm, 633nm and 405nm), 9 fluorescent detectors and high speed four-way cell sorting
• Beckman Coulter Cytomation MoFlo cell sorter with three lasers (excitation lines at 488nm, 405nm, and a Krypton tunable to 647nm and MLUV), 8 fluorescent detectors, high speed four-way bulk sorting and single cell deposition sorting
• BD LSRII SORP analyzer with three lasers (excitation lines at 488nm, 405nm and 647nm) and 16 fluorescent detectors
• BD LSRII analyzer with three lasers (excitation lines at 488nm, 405nm and 633nm) and 9 fluorescent detectors
• BD Canto II analyzer with three lasers (excitation lines at 488nm, 405nm, and 647nm) and 8 fluorescent detectors
• BD LSR I analyzer with three lasers (excitation lines at 488nm, 405nm and 633nm) and 6 fluorescent detectors.
• BD FACSort analyzer with two lasers (excitation lines at 488 nm and 635nm) and four fluorescent detectors
• Two BD FACScan analyzers with one laser excitation line at 488nm and three fluorescent detectors

Collaborations:
Part of the mission of the group is to give advice and experimental design suggestions to the investigators who use this facility. This advice includes trouble-shooting when results do not meet the expectation of the investigator. Staining protocols are kept on file and are provided to the investigator.

Publications:
Leeanansaksiri W, Wang H, Gooya JM, and Keller JR. IL-3 Induction of HLH protein Id1 in purified hematopoietic stem cells: A role for Id1 in myeloid versus lymphoid and erythroid cell fates. Molecular Aspects of Myeloid Stem Cell Development and Leukemia. 5: 13. Annapolis, Maryland, May 2003.

Suh H-C, Heath V, Holman M, Ortiz M, Gooya J, Renn K, Leeanansaksiri W, Friedman A, Johnson P, and Keller J. C/EBPalpha is required for common myeloid progenitor (CMP) differentiation and inhibits erythroid development. Molecular Aspects of Myeloid Stem Cell Development and Leukemia. 5: 49. Annapolis, Maryland, May 2003.

Liu P, Keller JR, Ortiz M, Tessarollo L, Rivka AR, Nakamura T, Jenkins NA, and Copeland NG. Bcl11a is essential for normal lymphoid development. Nat Immunol 4: 525-32, 2003.

Klein U, Tu Y, Stolovitzky GA, Keller JL, Haddad J Jr, Miljkovic V, Cattoretti G, Califano A, and Dalla-Favera R. Gene expression dynamics during germinal center transit in B cells. Ann N Y Acad Sci 987: 166-72, 2003.

Ortaldo JR and Young HA. Expression of IFN-gamma upon triggering of activating Ly49D NK receptors in vitro and in vivo: Costimulation with IL-12 or IL-18 overrides inhibitory receptors. J Immunol 170: 1763-9, 2003.

McVicar DW, Winkler-Pickett R, Taylor LS, Makrigiannis A, Bennett M, Anderson SK, and Ortaldo JR. Aberrant DAP12 signaling in the 129 strain of mice: Implications for the analysis of gene-targeted mice.
J Immunol 169: 1721-8, 2002.

Blanca IR, Bere EW, Young HA, and Ortaldo JR. Human B cell activation by autologous NK cells is regulated by CD40-CD40 ligand interaction: Role of memory B cells and CD5+ B cells. J Immunol 167: 6132-9, 2001.

Weiler SR, Gooya JM, Ortiz M, Tsai S, Collins SJ, and Keller JR. D3: A gene induced during myeloid cell differentiation of Lin1o c-Kit+ Sca-1+ progenitor cells. Blood 93: 527-36, 1999.

Papers in Preparation:
Saleh A, Hodge DL, Cho EH, Lockett SJ, Abshari M, and Anderson SK. Variegated gene expression: Identification of a programmable binary switch. In Progress, 2003.

Dermott JM, Gooya JG, Asefa B, Weiler SR, Smith MR, Copeland NG, Gilbert DJ, Jenkins NA, and Keller JR. Inhibition of growth by p205: A nuclear protein and putative tumor suppressor expressed during myeloid cell differentiation. In Progress, 2003.

Gooya JM, Klarmann K, Kelvin D, Lyman S, Ruscetti FW, Oppenheim J, and Keller JR. Interleukin-8 directly synergizes with steel factor to promote the growth of lineage-negative c-kit- positive progenitors. In Progress, 2003.