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The Flow Cytometry Core provides researchers with the ability to perform multi-parameter flow cytometric analysis and cell sorting utilizing state-of-the-art instrumentation. The mission of the Flow Cytometry Core is to offer expert technical assistance in analytical cytometry and cell-sorting, to train members of the Ordway Institute in practical flow cytometry, and to develop novel flow cytometric approaches and assays.

Flow cytometry is a laser-based technology that can rapidly analyze the biophysical, biochemical, and molecular properties of cells, most often through the use of fluorescent probes. Based on the fluorescent analysis of a mixed population of cells, a sub-population of cells that express a specific marker can be identified and physically sorted to a high purity.

The laboratory has the ability to perform many advanced flow cytometric assays, including:

• Detection of cell-surface and intracellular antigens
• Cell cycle, cell division and DNA ploidy analysis (BrdU for S phase and H3-p for M phase)
• Apoptosis assays (TUNEL, Caspases, Annexin V, mitochondria membrane potential and Sub-G1)
• DNA repair assay (gamma H2AX for dsb)
• Measurement of intracellular pH/calcium flux and efflux of fluorescent substrates
• Measurement of green fluorescent protein (GFP) and other fluorescent proteins
• High-throughput flow cytometric assays in 96-well plates
• Side population and CD marker assay for stem cells
• Direct cell cloning into microtiter tissue culture plates or onto microscope slides
• Aseptic sorting of living cell population (up to FOUR simultaneous collection tubes)
• Consulting (Design of experiments, analysis or conversion of data)

Laser	Wavelength (nm)	Commonly-used Fluorochromes
Coherent® Sapphire™ solid state	488 (blue)	FITC, GFP, Alexa488
		PE, PI, DsRed
		PE-Texas-Red
		PE-Texas-Red
		PE-Cy7
JDS Uniphase™ HeNe air cooled	633 (red)	APC, Cy5, Alexa 633, Alexa 647
		APC-Cy7
Point Source Violet solid state	407 (violet)	Alexa 405, Pacific Blue, DAPI, Hoechst 33342
		Alexa Fluor 430

The flagship instrument of the laboratory is the BD “FACS Aria” high-speed cell sorter. New-generation digital electronics allow high speed analysis and sorting (>20,000 cells per second). The FACS Aria optical system is configured with three excitation lasers and can detect 9 fluorescent parameters simultaneously. Additionally, the FACS Aria offers numerous sorting options which include 4-way sorting, sorting to microtiter plates or glass slides, and customizable settings to optimize sort yield and purity. The FACS Aria laser excitation wavelengths and partial list fluorochromes that can be utilized on the instrument are listed in the table.

For analytical purposes, the BD “LSR II” flow cytometer is available. The LSR II has digital electronics for increased speed, a 3-laser optical bench (see table), and can detect 8 fluorescent parameters (plus 2 physical parameters and a time parameter).

The laboratory also has a dedicated workstation for data analysis and the preparation of graphics. FACS Diva, WinMDI, and ModFit LT software packages are available for such work.

For further information about the Flow Cytometry Core’s capabilities, technical considerations in experimental design, fee schedule, or to make an appointment to use the instrumentation, please contact us.