Flow Cytometry Laboratory
The Flow Cytometry Lab is housed in the Animal Disease Biotechnology Facility (ADBF) Room 4075 and is operated by the WSU College of Veterinary Medicine.
The FC Lab has two analytical flow cytometers (FACS Calibur and FACSort) and one flow cytometer with sorting capability (FACS Vantage SE with fiber optics and Diva software operating system).
- The FACS Calibur and FACSort have two lasers and can analyze cells based on 6 separate parameters (side and forward light scatter, and detectors for 4 different fluorescent colors).
- The FACS Vantage SE is a high-speed cell sorter. It has two lasers and can analyze and sort based on 8 separate parameters (side and forward scatter and detectors for 6 different fluorescent colors). The FACS Vantage can simultaneously sort up to four populations of cells. It is also possible to sort one or more cell types aseptically into 6, 24, and 96 well tissue culture plates.
- There is a central data analysis software program (FCS Express) on the departmental network that can be accessed by signing up for its use.
Protocols and Services Available
- Immunophenotyping of peripheral blood cells and cell suspensions from tissues from a wide range of species. Flow cytometric phenotyping is used diagnostically to determine the cell type of malignancies, therapeutically to monitor cell populations, and in research to monitor cell populations following infection, disease course and immunization studies. Panels of monoclonal antibodies for use in specific species are available from the WSU Monoclonal Antibody Service Center.
Isolation of PBMC from peripheral blood
Labeling cells with monoclonal antibodies (mAbs) for Flow Cytometry
- Monitoring of harmful bacteria and protozoan species in water supplies.
- Isolation of a single cell type from a mixed cell population. Applications include production of specific T cell clones, enrichment of cells expressing a specific product such as a monoclonal antibody or GFP-tagged recombinant protein, and enriching the frequency of a rare cell type in a population.
- Determination of ploidy of cells using a DNA intercalating fluorescent marker. Applications include cell cycle analysis and detection of triploid or tetraploid cells.
- Monitoring of apoptosis.
- Detection of infection by intracellular pathogens.
- Cell viability. Use of propidium iodide to label dead cells
- Detection of cytokine or interleukin production by cells.
- Detection of intracellular antigens Intracellular detection of Foxp3
Panels of monoclonal antibodies are available for a variety of species. Antibodies can be purchased from the WSU Monoclonal Antibody Center.
Technical support is available to users of this facility. The FACS Calibur and FACSort are user-friendly flow cytometers that can be used by students, staff, and faculty without technical assistance after instruction on operation. Technical and operations support will be provided to researchers who wish to use the flow cytometers. Training will be provided to researchers who will require more than occasional use of these instruments. The FACS Vantage requires technical assistance at all times. Arrangements to schedule use of the cell sorter can be made by contacting Dr. Davis. Training in the use of the sorter will be provided to a designated technician or graduate student in laboratories requiring long-term heavy use of the sorter.
Authorized individuals have access to the core facility at all times during the week and weekends. Frequent users can obtain key badges. Also, a key to the room is available in Dr. Davis' laboratory for occasional users of the facility (Bustad Hall Room 324). Users may reserve the time they will be using the flow cytometers in advance. Call Dr. Davis' laboratory, 335-6058, to reserve time on the flow cytometers.
Hourly fees are charged for use of the cytometers. Contact Dr. Davis to inquire about fees.
Flow Cytometry Intensive Short Course
Duration: 2 weeks on site
1. Preparation of cells for labeling
2. Instruction on how to set up 1, 2, 3, and 4 color flow cytometry panels
3. Protocols for surface and intracellular labeling
4. Instruction on setting up an analytical flow cytometer, including compensation and collection of multiple colors
5. Data analysis using FCS Express
6. Instruction on setting up and using the FACS Vantage cell sorter
7. Starter set of monoclonal antibodies appropriate to the species of interest
WSU Monoclonal Antibody Center
Dr. William C. Davis
Veterinary Microbiology and Pathology
PO Box 647040
Washington State University
Pullman WA 99164-7040
Other sites of interest
Other sources include AbD Serotec and AbCam.
www.vetmed.wsu.edu/tkp The TKP program (taxonomic key program) is a program designed to assist investigators in the characterization of new mAbs specific for leukocyte differentiation molecules (LDM) in various species that are orthologues of LDM fully characterized in humans. Investigators can use the data base to compare the characteristics of the new mAb-defined molecule with the characteristics of known molecules, based on molecular weight, surface orientation, pattern of surface expression on one or more cell types, and molecular weight. A short description is provided in the data base. There is a link to a second database that provides information on existing mAbs that react with the respective CD molecules in one or more species. The antibody database can be accessed directly through the TKP for investigators attempting to find mAbs to the CD of interest.