Flow Cytometry Core

/Flow Cytometry Core
Flow Cytometry Core 2017-09-19T19:04:08+00:00

Flow Cytometry Core

The FCC supplies:

Research – The FCC supplies flow cytometry instrumentation, experimental protocols, technical support, maintenance, and administration for three instruments that are very intensively used in a variety of research endeavors. Much of the immunology, cell biology, and infectious disease work carried out at the CNPRC employs FCC services. The work of the FCC is guided by a Faculty Advisory Board to ensure that user and faculty needs are met.

Development and consultation – The FCC receives frequent requests for consultation on experiments that push the boundaries of current knowledge or are simply outside the comfort range of most investigators. Often such requests are focused on identification of certain cellular phenotypes in macaques. In these cases the FCC can often provide both guidance and preliminary data; alternatively, we can suggest a plan for developing appropriate panels and analytic approaches.

Training – The FCC trains all core users to ensure safe operation of the instruments and successful collection of data. Crucially, the FCC also provides in-person, on-site support during extended business hours. On-site support is provided so that user difficulties can be addressed quickly and the machines can remain continuously operational.

Service – Certain flow cytometry panels are required by Primate Medicine to provide the best possible animal care. These panels are offered as a service by the CNPRC Clinical Laboratory using FCC instruments.

Data collected by users are transferred from the cytometer by the user.  Data files remaining on the cytometer are backed up by the Flow Cytometry Core monthly and the files then deleted.

Data collected and/or analyzed by the Flow Cytometry Core are made available via LabKey.  You will receive login instructions from Dr. Hartigan-O’Connor.

The work of the FCC is guided by a Faculty Advisory Board to ensure that user and faculty needs are met.

The Faculty Advisory Board is charged with providing appropriate and objective advice and evaluation to the Core Lead, including feedback on issues important to users (e.g., machine uptime, service quality, scheduling) and advice on equipment needs.  The Faculty Advisory Board will also play an important role in guiding the FCC to important new methods, results, and funding opportunities.  The Board meets monthly to consider these issues and schedules additional meetings as needed.  Mr. Rourke attends all meetings.  The Faculty Advisory Board includes Dr. Hartigan-O’Connor, Dr. Nicole Baumgarth, Dr. Sungjin Kim, and two members who are external to the CNPRC: (i) Barbara Shacklett, Ph.D., is director of the UC Davis campus’s Flow Cytometry Shared Resource, and (ii) Jeffrey Milush, Ph.D., is director of the Core Immunology Laboratory in the Division of Experimental Medicine at UCSF, which provides cytometry services to the UCSF community.

Nicole Baumgarth, D.V.M., Ph.D. is Professor of Immunology at the Center for Comparative Medicine (adjacent to CNPRC) and in the Department of Pathology, Microbiology, and Immunology at UC Davis. Dr. Baumgarth studies the regulation of immune responses to infections and basic B cell biology. Her work on infections focuses on the regulation of B cell and B cell subset responses to two very different pathogens and corresponding immune responses in mouse models: acute, resolving infections with influenza virus and chronic, non-resolving infections with Borrelia burgdorferi, a bacterial spirochete and the causative agent of Lyme disease. By contrasting a successful with an unsuccessful B cell response, her group aims to uncover the immune regulatory mechanisms that shape the quality of B cell responses. Before joining UC Davis, Dr. Baumgarth was a Staff Scientist in the Herzenberg laboratory at Stanford University. While there, she worked with the Stanford FACS development group to develop 11+ parameter flow cytometry. She was one of the first to publish the use of fluorescent viral antigens as detection reagents for antigen-specific B cells by flow cytometry. She also designed and optimized the optical pathway of the 22-parameter, special-order Fortessa currently in use at the CNPRC and continues to provide the Core with calibration beads.

Sungjin Kim, Ph.D. is Associate Professor in the Department of Medical Microbiology and Immunology, UC Davis School of Medicine, and member of the Center for Comparative Medicine.  He is an internationally recognized expert in NK cell biology, particularly the flow cytometric definition of “memory” NK cell subsets.

Barbara L. Shacklett, Ph.D. is Professor of Medical Microbiology and Immunology, UC Davis School of Medicine. Her laboratory is recognized for developing and applying innovative approaches to study of mucosal immunity, primarily in humans but also in nonhuman primate models. Over the past 10 years, a major emphasis of her research has been to elucidate the relationship between mucosal T-cell responses and control of HIV infection. This work has included studies of HIV “Elite” Controllers, the role of mucosal regulatory T-cells, mucosal immune reconstitution in individuals on combination antiretroviral therapy, and mucosa-associated invariant T-cells (MAIT cells). Her group also studies the female reproductive tract, including hormonal regulation of innate and adaptive immune responses and the impact of menstrual cycle phases on host defense. Her laboratory has trained numerous graduate students, postdoctoral fellows, undergraduate trainees and domestic and international visiting scientists; her research to date has led to over 75 publications. Dr. Shacklett also serves as Scientific Director of the Flow Cytometry Shared Resource, part of both the UC Davis Cancer Center and School of Medicine.

Jeffrey M. Milush, Ph.D. is Director of the UCSF Core Immunology Laboratory (CIL) and UCSF Division of Experimental Medicine Flow Cytometry Core. His primary research interest is the use of flow cytometry and immunological assays to study pathogenesis of infectious and immune-mediated diseases. He has fourteen years of experience designing and directing laboratory studies in support of clinical research projects. From 2001 to 2016, CIL contributed to more than 100 peer-reviewed publications in areas including: T cell function and activation in relation to disease progression in HIV infection; neuroimmunology of HIV disease; HIV and aging; eradication of HIV-1 infection; and development of assays for studying immune cell subset phenotype and function. CIL has also developed a procedure for FACS sorting of endothelial cells from endothelial biopsies as well as an in situ hybridization assay to detect HIV-1 RNA expression in primary T cells from HIV-1-infected persons. Dr. Milush has extensive experience with designing and validating multiparameter flow cytometry panels for T cells, B cells and NK cells and has worked with clinical investigators to apply multiparameter flow cytometry analysis to large sets of fresh and cryopreserved samples.

  • flowClean is an algorithm to track subset frequency changes within a sample during acquisition, and flag time periods with fluorescence perturbations leading to the emergence of false populations. Aberrant time periods are reported as a new parameter and added to a revised data file, allowing users to easily review and exclude those events from further analysis.

User training at the Flow Cytometry Core involves reading flow core SOPs (startup, proper use, and shutdown); attending one or two initial teaching sessions with Tracy Rourke; and several sessions of supervised cytometer use, which can be extended as needed until the new user is comfortable.

Due to Mr. Rourke’s constant, on-site availability, most users can be trained within 72 hours of his or her first request.  One hour of initial training with only minimal data collection is provided, followed by one to several hours of use in the presence of Mr. Rourke or Ms. Spinner, depending on the user’s previous experience and aptitude.  Training can be also be provided to established researchers seeking to utilize flow cytometry as a new tool, as well as to pilot project recipients and visiting scientists using nonhuman primates for a wide range of research applications.

Training and consultation in data analysis are provided by Dr. Hartigan-O’Connor.