Frederick CCR Flow Cytometry Core
Laboratory of Experimental Immunology (LEI)

Comprehensive support for studies of cell sorting, cell cycle, apoptosis, and cell ploidy

Mission: Flow cytometry (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 two related programs. First, basic research support is provided to members of the Frederick-CCR and, on a limited basis, to investigators elsewhere at NIH. The laboratory staff also participates in the development of flow cytometry applications and resources for use in immunological research. Resource development in the lab includes advances in instrumentation, reagents, methodology and computer hardware/software. This laboratory is contract-operated with direction provided by Dr. John Ortaldo.

Access: Lab/Program and Dedicated

Staff:
CCR Core Supervisor - Kathleen Noer, BS noer@ncifcrf.gov 301-846-5811
High-Speed Sorter Core Supervisor - Mehrnoosh Abshari AbshariM@ncifcrf.gov 301-846-1549
Support Staff - Roberta Matthai, BS rmatthai@ncifcrf.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:

• Fluorescence protein sorting such as: e-BFP, e-CFP, e-GFP, e-YFP, Ds-Red
• Cell cycle sorting
• Multicolor sorting of up to 5 colors
• Cell division sorting by Hoechst 33342 and CFDA-SE
• Simultaneous detection of cyan and yellow fluorescence protein and their sorting
• Immunophenotyping with 6-color analysis
• Cell-cycle and cell-ploidy analysis
• Calcium mobilization analysis
• Apoptosis analysis
• Immunophenotyping of transgenic/knockout mice

Instrumentation:
Dako-Cytomation MoFlow: The state-of-the-art MoFlo® High-Performance Cell Sorter is a modular, custom-configured instrument that provides a complete solution to all of your sorting needs. The instrument is equipped with 3 water-cooled coherent lasers:

1. I-70c tunable Argon laser operated at 488 single-line primary laser
2. I-304c tunable Argon laser operated at UV multi-line 335-365 nm
3. I-302c tunable Krypton laser operated at violet (407 & 413) and red (647) single lines

This instrument has the capability of detecting 10 parameters, FSC and SSC, and 8 fluorochromes simultaneously using as many as three laser lines.

MoFlo can sort up to 4 different populations simultaneously. It has the capability of sorting up to 50,000 cells per second. Depending on the sample, the purity of the sorted cells is above 90% with a recovery of 70-80%. The Moflo can sort into a variety of capture vessels. In addition to test tubes typically used for bulk sorts, the Cyclone on the MoFlo can deposit a variable number of cells into a variety of catch vessels; 6-, 12-, 24-, 48-, 96-, and 384-well cluster plates as well as onto slides. It is also possible to sort cells directly onto filters or nitrocellulose membranes.

Becton Dickinson LSR: A bench-top analyzer with Argon, HeNe and HeCad lasers and 8 acquisition parameters

Becton Dickinson FACScan: A bench-top analyzer with an Argon laser and up to 3-color analysis

Becton Dickinson FACSTAR^PLUS: A conventional sorter with two Argon lasers, one of which has UV capability

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.