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Flow Cytometry Core (SOM/CVD)

Scientific objectives

The primary goal of the CVD flow cytometry/cell sorting Core Laboratory is to ensure that University of Maryland investigators whose research projects require the use of a flow cytometer have access to such instrumentation. As this equipment is very expensive and very time-consuming to become trained on, it is much more efficient to have a facility with a dedicated operator(s) to run the equipment. Established in 1991, this facility has state-of-the-art equipment and a highly-trained and experienced staff.

Measurements/Services

  1. Characterization of cell subpopulations by multichromatic conventional flow cytometry (up to 14 simultaneous fluorochromes plus forward and side light scatter parameters) or mass cytometry (once installed in early 2013 we expect to be able to measure 30 or more simultaneous parameters). These are cutting-edge technologies.
  2. Measurement of intracellular cytokine levels and other molecules and determination of the expression of cytokine and chemokine receptors in defined cell populations by multichromatic flow cytometry or mass cytometry.
  3. Measurement of serum/supernatant cytokine levels using commercially available bead kits, such as the BD Pharmingen cytometry bead array (CBA) assay kit.
  4. Cell cycle analysis as determined by propidium iodide or 7-AAD staining and measurement of cell proliferation by CFSE, PCNA, BrdU and Ki67 staining.
  5. Determination of apoptosis in individual cells as measured by TUNEL staining, simultaneous staining with Annexin V and propidium iodide, and subG0/G1 peak analysis using DNA dyes such as propidium iodide or 7-AAD.
  6. Measurement of expression of green fluorescence protein (GFP) in transiently or stably transfected eukaryotic and prokaryotic cells.
  7. Physical isolation of cell subpopulations by flow cytometric cell sorting (2- and 4-way) based on expression of GFP and/or other markers defined by multichromatic monoclonal antibody staining.

Instrumentation

  • DakoCytomation MoFlo flow cytometer and cell sorter with 4-way sorting and up to 12 parameters (capable of 10 colors plus forward and side scatter; this is state of the art).
  • BD Biosciences LSR II flow cytometer with 16 parameters (capable of 14 colors plus forward and side scatter; this is state of the art).
  • CyTOF Mass Cytometer (expected in early 2013) with the capability of measuring over 30 parameters simultaneously in a single tube. This cutting-edge technology is based on the detection of cells labeled with stable isotope-bound monoclonal antibodies by mass spec.

Personnel

Marcelo Sztein, M.D.

Associate Director for Immunologic Studies, Leader, Immunology Group and Chief of the Cellular Immunology Section and Flow Cytometry Core Laboratory of the Center for Vaccine Development (CVD). Holds an appointment as tenured Professor, Division of Infectious Diseases and Tropical Pediatrics, Department of Pediatrics and secondary appointments in the Departments of Medicine and Microbiology and Immunology Has 26 years of experience in flow cytometry and has been director of the CVD’s Flow Cytometry Core Laboratory since its inception in 1991. Has over 35 years of experience in the study of molecules involved in the regulation of cellular, humoral, innate and mucosal immunity, with particular emphasis on the understanding of the mechanisms underlying the generation of immune responses to infectious agents in humans and animal models.

Regina Harley, M.S.

Has been Lab Supervisor of the CVD Flow Cytometry Core Laboratory since November 1999. Lab Supervisor of the University of Rochester Cancer Center Flow Cytometry Facility for 10 years prior. Has had professional training on all of the flow cytometers in the CVD Core Laboratory as well as others. Has extensive experience in all of the services listed above, including multichromatic flow cytometry, cell sorting (2- and 4-way), CBA assays, cell cycle analysis, measurement of cell proliferation, determination of apoptosis, measurement of expression of green fluorescence protein (GFP), etc.

Cathy Storer, B.S.

Has over 20 years of experience in analytical flow cytometry, including complex multicolor analysis, cytometry bead arrays (CBA), proliferation, etc.

Pricing

All flow cytometric sample analysis and cell sorting is done by Core Laboratory personnel and is charged at a rate of $100.00/hour, rounded up to the nearest half hour. Sorting has an additional flat 2-hour set-up charge ($200.00).

A final decision on the CyTOF Mass Cytometer charge rate has not been made; however it is likely to be ~$150.00/hour.

Laboratory Policies

The "Rules and Regulations" form (Revision May 10, 2005) is available at the CVD Flow Cytometry Core Laboratory.

Contact

Marcelo Sztein, MD
HSF I, Room 456
(410) 706-5328