Cellular Immunity and Flow Cytometry Subcore

The mission of the flow cytometry subcore is to support all phases of experimental studies using flow cytometry including experimental design, staining panel design, laboratory procedures, collection of samples on the instrument, analysis of data and interpretation of results.

The flow cytometry core provides support for experiments utilizing flow cytometry. These can be simpler experiments using one to three colors or cutting-edge experiments using up to 18 colors. Support can be provided for all phases of these studies, including experimental design, staining panel design, laboratory procedures, collection of samples on the instrument, analysis of data and interpretation of results. Our aim is to serve as a resource to promote research in HIV and HIV-related diseases and we can provide multiple levels of support ranging from answering questions by email or phone, to providing individual training in laboratory procedures and also providing samples of specialized fluorochrome-conjugated antibody reagents.

The specialized cellular immunoassay aims to train individuals in state-of-the-art functional immunoassays. We can aid in assay design, optimization and validation of new assays as well as provide training for: T cell functional assays (measures of proliferation, cytotoxicity, cytokine secretion, viral suppression, etc) and NK functional assays (cytotoxicity, cytokine secretion, viral suppression, ADCVI).

We can also perform small or pilot experiments that may, for example, be necessary for a grant application. Please feel free to contact us.

Assays for Cell Proliferation

CFSE is commonly used to measure cell proliferation by flow cytometry. We have standardized the assay and posted a protocol on the resources page.

For more information on how to access this service, contact Stephen De Rosa at sderosa@fredhutch.org.

Cell Sorting

We can provide assistance in designing experiments to sort cell populations of interest. We can perform pilot sorting experiments. For more extensive sorting experiments, we can help you plan for experiments using sorter instruments located in the shared flow facilities nearby.

Fred Hutchinson Shared Resources: Flow Cytometry

University of Washington Department of Immunology Resources

For more information on how to access this service, contact Stephen De Rosa at sderosa at fredhutch.org.

Cytokine Bead Array

These assays (such as Luminex) measure concentrations of multiple analytes simultaneously. We use these assays for measurement in serum/plasma and in supernatants from PBMC stimulated in culture.

For more information on how to access this service, contact cfar@uw.edu

Experiment Design

When designing a flow cytometry experiment, it is often useful to take advantage of prior experience in terms of combinations of colors and markers that work well. We have worked with markers to examine most leukocyte cell types in blood. In addition to cell phenotyping, we have experience measuring cell function using the intracellular cytokine staining assay. We hope that our expertise can help streamline the design of your experiments.

For more information on how to access this service, contact Stephen De Rosa at sderosa at fredhutch.org.

Fee-for-Service Performance of Assays

If you are interested in utilizing our fee-for-service, please contact cfarinfo@uw.edu.

Flow Cytometry Consultations

Please contact us for any questions concerning flow cytometry or cellular immunology and we will try to help or direct you to someone with appropriate expertise.

For more information on how to access this service, contact Stephen De Rosa at sderosa@fredhutch.org.

Flow Cytometry Training

We provide all levels of training from basic to advanced. We often offer workshops for more formal training opportunities, but we offer individual training by phone, email, or in person.

Here is a link to the slides used for our “Introduction to Flow Cytometry” Workshop, and click here to view the slides from the Flow Workshop covering more advanced concepts and methods.

For more information on how to access this service, contact Stephen De Rosa at sderosa@fredhutch.org.

NK Cell Functional Assays

NK cell immunophenotyping: can include inhibitory and activating receptors for NK cells.
Viral Suppression Assay (VSA): Measures the ability of NK cells to inhibit viral replication in autologous CD4 cells. This inhibition measures a combination of both direct cytotoxicity and chemokine/cytokine secretion.
Cytotoxicity Assay: Measures the direct killing activity of NK cells when they encounter HIV-infected targets. This is a flow-based assay.
Antibody-dependent Cell-mediated Viral Inhibition (ADCVI): Measures the ability of HIV-specific antibodies to mediate killing of HIV-infected cells via NK cell activation. Assay can be performed with autologous or heterologous NK cells.
For more information on how to access these services, contact Ana Gervassi at ana.gervassi@cidresearch.org

T Cell Functional Assays

ELISpot: Measures the frequency of IFN-g-secreting T cells in peripheral blood mononuclear cells (PBMC). Is more sensitive than intracellular cytokine staining (ICS).
ICS: Measures the frequency of antigen-specific T cells secreting numerous cytokines/chemokines (e.g. IFN-g, TNF-a, IL-2). Also determines phenotype of responding T cells (CD4 or CD8).
Sorting of HIV-infected and/or TB-infected cells
MHC Restriction: Determines which HLA allele is used to restrict a specific T cell response.
Viral Suppression Assay (VSA): Measures the ability of HIV-specific T cells to inhibit viral replication in autologous CD4 cells. Can be performed with bulk T cells or with T cell lines/clones specific for certain HIV epitopes.
Cytotoxicity Assays: Measures the direct killing activity of HIV-specific T cells. This is a flow-based assay.
Proliferation Assays: Measures ability of T cells (both CD4 and CD8) to proliferate in response to HIV antigens/epitopes. This is a flow-based assay using CFSE.
Treg Suppression Assay: Measures the suppressive effects of Tregs on HIV-specific effector cells (both CD4 and CD8). Can measure suppressive effects of Tregs on either cytokine secretion or proliferation of effectors.
siRNA transfection of primary T cells (including Tregs).

For more information on how to access these services, contact Stephen de Rosa at sderosa@fredhutch.org

content TBD

Stephen De Rosa
Subcore Director
Email: sderosa at fredhutch.org
Telephone: 206-667-1681206-667-1681

Stephen De Rosa is available for consultation by email, phone or in person. The laboratory is located at Fred Hutchinson Cancer Research Center at the 1100 Eastlake Building.