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Molecular and Cellular Immunology Core

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As the sole resource for flow cytometry, cell sorting and state-of-the-art immunological services in the western-most IDeA state, the Molecular and Cellular Immunology Core, through COBRE support, has accelerated research productivity, in terms of publications and extramural funding. Emphasis has also been applied to developing new or customized immunological methods for COBRE Investigators and other Core users. In addition, regularly scheduled training sessions are held to enrich the educational and mentoring experience for COBRE Investigators and other faculty and students across the university and broader research community. By centralizing immunological services and resources to this Core, COBRE Investigators and other researchers will be more efficiently served and the use of expensive equipment will be maximized.

Objective

Enhance the infrastructure for the Molecular and Cellular Immunology Core, in support of hypothesis-driven research projects that seek to gain new knowledge about emerging infectious diseases.

Specific Aim 1

  • Enhance and streamline core operations.

Specific Aim 2

  • Grow and diversify user base, capability, capacity and reach.

Specific Aim 3

  • Strengthen core infrastructure.
People
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George S.N. Hui, Ph.D.

Director, Molecular and Cellular Immunology Core
Professor
Department of Tropical Medicine, Medical Microbiology and Pharmacology
John A. Burns School of Medicine


Email: ghui [at] hawaii.edu
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Alexandra Gurary, Ph.D.

Associate Director, Molecular and Cellular Immunology Core


Email: gurary [at] hawaii.edu

Services/Assays

Core Services:

  • Flow Cytometry: Analysis and Sorting
  • Cell Counting, Size, and Viability
  • Multiplex Bead Assays: Luminex and CBA Platforms for DNA, Protein, and Antibodies Assays
  • Immunospot Plate Reading: ELISpot and FRNT
  • Cell Irradiation


Flow Cytometry Assays:

  • Cell Phenotyping
  • Cell Cycle with Live or Fixed Cells
  • Apoptosis
  • FRET
  • Calcium Flux
  • Phosphorylation
  • Intracellular Cytokine and Protein Detection
  • Fluorescent Protein Expression
  • Cell Population Isolation
  • Single Cell Sorting
  • Dye Dilution Assays
  • Proliferation

Training opportunities

Experienced staff will provide hands-on training to investigators in conducting IBC/IACUC/CHS approved MCI research protocols. Please refer to MCI training document to help you get started on the process for obtaining clearance to work in the JABSOM Molecular and Cellular Immunology Core Facility.


Please contact Alexandra Gurary with any questions regarding flow cytometry, new applications or our facility at gurary[at]hawaii.edu, or you can give us a call at 808-692-1794 to discuss how the MCI core can complement your research.

Equipment
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Protocols/Procedures

As required by the COBRE EAC, we require that users submit a Service Request Form once a year for each research project, and a Sample Description Form for every appointment made with flow core operator, Alex Gurary.


Please fill out the service request and sample description form and email it to the Molecular and Cellular Immunology Core Facility.


All forms must be opened and completed with Adobe Reader.

Publications

Citations in Publications

Use of this core facility should be acknowledged in publications, abstracts, posters and oral presentations. The suggested verbiage is:


“Some of the services for this research were provided by the Molecular and Cellular Immunology Core, which is supported in part by grant P30GM114737 from the Centers of Biomedical Research Excellence (COBRE) program of the National Institute of General Medical Sciences, a component of the National Institutes of Health.”


2011-2012


2010-2011


2009-2010

Flow Cytometry Basics

GUIDE TO FLOW CYTOMETRY AND ASSAYS

The acronym FACS (Fluorescence Activated Cell Sorting) and flow cytometry are used interchangeably. FACS is a powerful method used to study and purify cells. FACS has a wide application in immunology and cell biology and other fields of biology.


Individual cells held in a thin stream of fluid are passed through one or more laser beams cause light to scatter and fluorescent dyes to emit light at various frequencies. Photomultiplier tubes (PMT) convert light to electrical signals and cell data is collected. Cell sub-populations are identified and sorted at high purity (~100%). FACS instruments generate three types of data:


- Forward Scatter (FSc) - Approximate cell size
- Fluorescent Labeling - Used to investigate cell structure and function
- Side or Orthogonal Scatter (SSc) - Cell complexity or granularity


Scatter
Forward and side scatter are used for preliminary identification of cells. In a peripheral blood sample, lymphocyte, monocyte and granulocyte populations can be defined on the basis of forward and side scatter. Forward and side scatter are used to exclude debris and dead cells.


Fluorescence
Fluorescent labeling allows investigation of cell structure and function. Cell auto fluorescence is generated by labeling cell structures with fluorescent dyes. FACS collect fluorescence signals in one to several channels corresponding to different laser excitation and fluorescence emission wavelength.


Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein and phycoerythrin. This method is often used to label molecules on the cell surface, but antibodies can be directed at targets in cytoplasm.


In direct immunofluorescence an antibody to a molecule is directly conjugated to a fluorescent dye (such as lymphocyte surface marker CD4). Cells are stained in one step.


In indirect immunofluorescence the primary antibody is not labeled. A second fluorescently conjugated antibody is added which is specific for the first antibody. For example, if the anti-CD4 antibody was a mouse IgG then the second antibody could beat rat antibody raised against mouse IgG. Immunofluorescence examples:


- Quantifying CD4 and CD8 subsets of T lymphocytes
- Intracellular cytokine staining


The use of Flow Cytometry can be divided into two broad categories, analysis and cell sorting.


Analysis
The ability of flow cytometers to evaluate cells at an extremely rapid rate (e.g. up to 20,000 events per second) makes this technology ideally suited for the reliable and accurate quantitative analysis of selected physical properties of cells of interest. The sensitivity of these instruments for detecting the presence of molecules expressed at low levels is impressive; given high quality cell preparations and reagents, as few as 50 molecules per cell may be detected.


Cell Sorting
One of the properties of the larger flow cytometers is the ability to electronically deflect cells with preset, defined properties into a separate collection tube. For cell purification, flow cytometry is especially well suited for applications requiring high purity. Because multiple fluorochromes (e.g. up to eight distinct fluorescent probes reacting with different cell associated molecules) can be assessed simultaneously, cell sorting by flow cytometry can separate complex mixtures of cells on the basis of multiple marker expression.


DNA Staining
FACS is used to study DNA cell content. Propidium iodide (PI) and Hoechst dyes bind to DNA and become fluorescent. PI cannot enter live cells and is included in immunofluorescent staining protocols to identify dead cells. Some Hoechst dyes can enter live cells. DNA staining can be used to study the cell division cycle. Relative DNA content shows the proportion of cells in G1, G2 and S phases. Apoptotic cells show characteristic smear on DNA staining. DNA staining examples:


- Studying changes in cell cycle in mutant cell
- Measuring apoptosis in cells after irradiation

Calcium Flux and Other Metabolic Studies

FACS can be used to investigate cell biology. Calcium flux can be measured using Indo-1 markers. This can be combined with immunofluorescent stain. For example, identify T cell subpopulations by immunofluorescence and measure calcium flux in response to an activating signal. Rhodamine-123 stains mitochondrial membranes is used to measure cellular activation. Rhodamine-123 is rapidly pumped out of some cells (for example hematopoietic stem cells).


CFSE binds to cell membranes and equally distributes when cells divide. Cell divisions in a period of time can then be counted. For example, labeling a population of cells with CFSE in vitro and reintroduce them in vivo. After a few days, the cells could be sampled and the amount of division measured. For example: Metabolic characteristics such as calcium flux, mitochondrial activity, pH, and free radical production can be measured in live cell populations in real time.

Gene Expression and Transfection

FACS is used to measure gene expression in cells transfected with recombinant DNA. This is achieved directly by labeling the protein product, or indirectly by using a reporter gene in the construct. Direct immunofluorescent labeling allows quantification of the product, and is suitable for relatively abundant proteins expressed on the cell surface. Indirect detection by reporter genes allows detection of transfectants at lower levels which cannot be detected easily by immunofluorescence. Examples of reporter genes are beta galactosidase and Green Fluorescent Protein (GFP). Beta galactosidase activity can be detected by FACS using fluorogenic substrates such as fluorescein digalactoside (FDG). FDG is introduced into cells by hypotonic shock and is cleaved by the enzyme to generate a fluorescent product trapped within the cell. One enzyme can generate a large amount of fluorescent product.


Cells expressing GFP constructs will fluoresce without addition of a substrate. GPF mutants are available which have different excitation frequencies, but emit fluorescence in the same channel. In a two-laser FACS, it is possible to distinguish cells which are excited by different lasers and assay two transfections at the same time.


- Expression of proteins can be measured directly by FACS.
- Transfection efficiency can be accurately determined.
- Transfection assays can be combined with staining and sorting for other markers.
- Transfected cells can be purified for analysis or use.

Dyes

Fluorescent dyes allow analyses of simultaneous parameters that can refine cell subpopulations. The fluorescent dyes that you can use will depend upon instrument used.


When different fluorescent dyes are used, signal spillover can occur between fluorescence channels. This needs to be corrected by setting compensation.


Setting correct compensation is important for obtaining accurate results when using multiple colors. In our facility we recommend use of compensation beads, when possible. Please contact Alexandra Gurary at gurary[at]hawaii.edu for further information.
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Links

Contact Us


Please contact George Hui or Alexandra Gurary with any questions regarding flow cytometry and new applications or questions about the Molecular and Cellular Immunology Core.

Director:

George Hui
Email: ghui[at]hawaii.edu

Core Supervisor:

Alexandra Gurary
Phone: 808-692-1794
Email: gurary[at]hawaii.edu