Office for Research and Graduate Studies (ORAGS)

Flow Cytometry—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.

Staff:

Director: Marcelo B. Sztein, M.D. HSF1 480 (410) 706-2345
Supervisor:Regina Harley, M.S. HSF1 456 (410) 706-7376
Back-up Staff: Steve Gallegos, B.S. and Jeffrey Floyd, B.S. 
Fax: (410) 706-6205
Email:  msztein@medicine.umaryland.edu  or  rharley@medicine.umaryland.edu
Location: Health Science Facility 1, Room 456
Website: http://medschool.umaryland.edu/orags/flowlab.asp
Hours: Monday - Friday, 9:00 am – 5:00 pm, excluding official university holidays

Measurements/Services:

1. Characterization of cell subpopulations by multichromatic flow cytometry (up to 10 simultaneous fluorochromes plus forward and side light scatter parameters).  This is a state-of-the-art flow cytometry system.

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.

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.

Experiments should be scheduled as far in advance as possible. Generally it takes about two weeks to be fit into the schedule.

Instrumentation:

Beckman Coulter Epics Elite ESP flow cytometer and cell sorter
DakoCytomation MoFlo flow cytometer and cell sorter with 4-way sorting and 10 PMTs (this is state-of-the-art)

Pricing (as of 6/15/01):

All sample analysis and cell sorting is done by Core Laboratory personnel and is charged at a rate of $75.00/hour, rounded up to the nearest half hour. Sorting has an additional flat 2-hour set-up charge ($150.00).

Staff:

Marcelo Sztein, M.D.

Leader, CVD Immunology Group and Chief of the Cellular Immunology Section and Flow Cytometry Core Laboratory.  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 23 years experience in flow cytometry and has been director of the CVD’s Flow Cytometry Core Laboratory since its inception in 1991. 
Has over 30 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.

Steve Gallegos, B.S. - CBA Assay and measurement of expression of green fluorescence protein (GFP)

Jeffrey Floyd, B.S. - preparation of samples for CBA assays.

Laboratory Policies:

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

Facility Advisory Committee members:
A Facility Advisory Committee has not been established.
 
Satisfied Customers:

Department of Endocrinology
Dr. Alan Shuldiner, Professor and Head, Div. of Endocrinology, Diabetes & Nutrition
Dr. John McLenithan, Assistant Professor
Department of Medicine, Div. of Geographic Medicine
Dr. Alan Cross, Professor
Dr. Christopher Plowe, Associate Professor
Dr. Kirsten Lyke, Assistant Professor
Dr. Eileen Barry, Research Associate Professor
Dr. James Galen, Research Associate Professor
Department of Medicine, Div. of Infectious Diseases
Dr. Simeon Goldblum, Professor, Section Chief of Infectious Disease at VAMC

Department of Microbiology and Immunology
Dr. Stefanie Vogel, Professor
Dr. Martin Flajnik, Professor
Dr. James Kaper, Professor
Dr. Abdu Azad, Professor
Dr. John Sacci, Assistant Professor 

Department of Pediatrics
Dr. Alessio Fasano, Professor and Head, Div. of Pediatric Gastroenterology & Nutrition
Dr. Karen Kotloff, Professor, Div. of Infectious Diseases and Tropical Pediatrics
Dr. James Nataro, Professor, Div. of Infectious Diseases and Tropical Pediatrics
Dr. Marcela Pasetti, Assistant Professor, Div. of Infectious Diseases and Tropical Pediatrics