Creighton University

The Flow Cytometry Core Facility, based in the Department of Medical Microbiology and Immunology, and located in room 525 of Criss II, provides equipment and services to identify, quantify, analyze, and isolate cells in complex mixtures (like blood, for example). The facility is equipped with two state-of-the-art flow cytometers: a BD FACSCalibur for 4-color analysis and a recently acquired BD FACSAria for high-speed sorting and multicolor analysis using three different lasers (350 nm [UV], 488 nm, and 633 nm) which allows for rapid purification of specific cell populations.

In addition to the computers that run these 2 instruments, the facility has a PowerMac G4 workstation setup as a data server and data analysis workstation.  All computers in the facility have CD-R/RW and DVD-R/RW drives, Iomega zip drives, floppy drives, and are networked to black & white and color laser printers.  All data generated in the facility is routinely backed up onto an additional 300 GB external hard drive and to CD/DVD disks.

Along with the 2 flow cytometers, the facility contains two magnetic cell separation units (a VarioMACS and a QuadMACS) from Miltenyi Biotech.  Using monoclonal antibodies and magnetic beads, these units allow for the rapid purification or enrichment of specific cell populations for further purification on the FACSAria, analysis or cell culture.

The Core also houses a model Z1 Coulter Counter for performing cell counts, an IEC Centra 8R refrigerated centrifuge, a Nikon E-400 microscope, and a Digital Blue digital microscope.

Application: Cell Analysis and Cell Sorting

In this experiment, two populations of B cells are being sorted from transgenic mice.  The two B cell populations are identified by staining with fluorochrome-labeled antibodies against two different B cell markers called CD19 and B220. The left-most panel shows the staining pattern of CD19-labeled splenocytes before sorting. Regions or "gates" are drawn around the two B cell populations to be sorted, called B220lo and B220hi. The middle and right-most panels show the purity of the B220lo and B22hi populations, respectively, after sorting.

In addition to sorting eukaryotic cells, the FACSAria is equipped to analyze and sort prokaryotic cells. Flow cytometry can also be used to analyze cell apoptosis and cell cycle status, measure levels of intracellular and secreted proteins (cytokines, chemokines, immunoglobulin, etc), and evaluate activation of cell signaling pathways (using BD PhosFlow Reagents).

Please contact Core personnel at 402-280-1841 if you have interest in performing these assays, or are interested in developing your own assays using flow cytometry. They will happy to help you in the design and optimization of your experiment.   Further information on facility policies, protocols and techniques can be obtained on the web site listed above.

Key Personnel

Patrick C. Swanson, Ph.D., Director     pswanson@creighton.edu
Greg Perry, Ph.D., Technical Director    cytometry@creighton.edu

Ongoing/Current Projects

• Cell cycle kinetics and apoptosis in UV-induced skin epithelial cell damage.
• Cell cycle kinetics and apoptosis in carcinogenesis.
• Dendritic cell populations and their functions in allergic responses.
• Regulatory T cell populations and their functions in allergic responses.
• The effects of oxidants and anti-oxidants on vascular smooth muscle cells and its role in coronary artery disease.
• The interplay between antigen receptor signaling and antigen receptor gene rearrangement in lymphocyte differentiation and tolerance.
• The effects of alcohol and smoking on polymorphonuclear phagocytes in host defense against bacterial pneumonia.
• Cell cycle kinetics and apoptosis in embryogenesis.
• Role of cytokines and immunoregulatory cells in autoimmune disease.
• The relationship between lipoprotein particles, oxidative stress, platelet reactivity and degree of endothelial function in African American smokers and non-smokers.
• Analysis of RNA interference (RNAi) mediated by siRNA-generating ribozymes.
• Role of bacterial efflux pumps in antibiotic resistance and antimicrobial therapy.

Ongoing/Current Projects

• Cell cycle kinetics and apoptosis in UV-induced skin epithelial cell damage.
• Cell cycle kinetics and apoptosis in carcinogenesis.
• Dendritic cell populations and their functions in allergic responses.
• Regulatory T cell populations and their functions in allergic responses.
• The effects of oxidants and anti-oxidants on vascular smooth muscle cells and its role in coronary artery disease.
• The interplay between antigen receptor signaling and antigen receptor gene rearrangement in lymphocyte differentiation and tolerance.
• The effects of alcohol and smoking on polymorphonuclear phagocytes in host defense against bacterial pneumonia.
• Cell cycle kinetics and apoptosis in embryogenesis.
• Role of cytokines and immunoregulatory cells in autoimmune disease.
• The relationship between lipoprotein particles, oxidative stress, platelet reactivity and degree of endothelial function in African American smokers and non-smokers.
• Analysis of RNA interference (RNAi) mediated by siRNA-generating ribozymes.
• Role of bacterial efflux pumps in antibiotic resistance and antimicrobial therapy.