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Immunology Core: Flow Cytometry Lab

Provide comprehensive cell sorting and phenotypic flow cytometry assay services

The Flow Cytometry Sorting Component of the Duke CFAR Immunology Core houses the only flow cytometry sorters at Duke capable of sorting live, unfixed human or animal derived cells, infected with HIV-1 or other pathogens requiring BSL2-enhanced or BSL3 containment and work practices. This capacity to sort live, unfixed HIV-1 infected cell subpopulations has broadened the scope of several ongoing, NIH-funded, AIDS research studies at Duke (e.g. CHAVI-ID).

The Analytical Flow Cytometry Component is the only Duke Shared Resource offering full service GCLP-compliant flow cytometry assays, as well as operator-assisted or investigator acquisition. The Analytical Flow Cytometry Component specializes in the development of highly standardized/validated polychromatic flow cytometry (PFC) assays for immune monitoring and serves as an A2LA-Accredited Proficiency Testing Provider for the NIAID/DAIDS-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program. Additional services include consultation (provided at no charge), assay development, custom polychromatic panel design, sample preparation, data analysis and archival, graphics and writing support (letters of intent, funding applications, publications, and presentations).

Provide biobanking services for plasma and viably preserved peripheral blood mononuclear cells (PBMC)

The Analytical Flow Cytometry Component offers sample pickup, transport, PBMC processing, isolation, cryopreservation, and storage for PBMC and plasma.

Provide scientific training and mentoring to Duke CFAR investigators and Early Stage Investigators (ESI) and lead efforts to establish collaborative ties across the CFAR network

The Analytical Flow Cytometry Component offers one-on-one training, hosts an annual week-long Duke CFAR Flow Cytometry Wet-Workshop , and coordinates the inter-CFAR Cytometry Interest Group (CIG), including sponsoring the annual inter-CFAR CIG Meeting as part of the Annual CFAR Director’s Meetings. In addition, the Flow Cytometry Component has established scientific collaborations with key leaders in the flow cytometry industry and, through these collaborations, routinely offers the latest in cutting-edge technology to Duke CFAR investigators as well as other members of the Duke community.

Services Offered

Consultation Services

The Flow Cytometry Component offers consultation services for flow cytometry assays at no charge to Duke CFAR Investigators. Consultation services include cost estimations, recommendations on sample collection, labeling, handling, shipping, storage, processing, cryopreservation, thawing, stimulation, staining, acquisition, analysis, and reporting. We also provide assistance in the design and preparation of grant/contract applications as well as manuscript preparation. We encourage investigators to take advantage of our free Consultation Services by speaking with us about experimental design and goals prior to initiating projects.

BSL2/BSL3 Cell Sorting

The Sorting Component of the Immunology Core houses the only flow cytometry sorters at Duke capable of sorting live, unfixed human or animal derived cells, infected with HIV-1 or other pathogens requiring BSL2-enhanced or BSL3 containment and work practices. This capacity to sort live, unfixed HIV-1 infected cell subpopulations has broadened the scope of several ongoing, NIH-funded, AIDS research studies at Duke (e.g. CHAVI-ID). The BSL2/BSL-3 Sorters are operated by well-trained operators with established procedures approved by Duke’s Biological Safety Division.

For more information regarding BSL2/BSL3 sorting services currently available to Duke CFAR Investigators, please contact Dr. M. Anthony (Tony) Moody.

Polychromatic Analytical Flow Cytometry

Shared Resource Rates

CFAR Investigators may elect to undergo instrumentation training for independent use or may elect to utilize operator-assisted acquisition services at established Shared Resource rates. The Core also offers access to an automated cell counters, refrigerators, freezers, water baths and incubators for those investigator’s who wish to use Core lab space to complete their flow cytometry experiments with supervision or assistance by Core staff.

Details are provided below for specific services offered by the Analytical Flow Cytometry Component. For more information regarding Analytical Flow Cytometry Component services currently available to Duke CFAR Investigators, please contact Dr. David Murdoch.

  • Core Operator Assistance
    The Analytical Flow Cytometry Component maintains a team of highly qualified Core Operators and offer full support of flow cytometry assays, from conception and design through data analysis and reporting. Core Operators are available to assist Investigators with sample labeling, handling, shipping, storage, processing, cryopreservation, thawing, stimulation, staining, acquisition, analysis, and reporting.
     
  • Flow Cytometry Analysis Workstations
    The Analytical Flow Cytometry Component maintains Macintosh and PC workstations that may be used free-of-charge by Duke CFAR Investigators to analyze data collected in the Duke Immunology Core.
     
  • GCLP-compliant flow-based assays
    The Analytical Flow Cytometry Component is uniquely qualified to offer flow cytometry services in a fully GCLP-compliant environment. The Analytical Flow Cytometry Component has a long-standing history of performing highly standardized/validated flow-based assays in support of HIV clinical trials, working with extra-mural networks to optimize/standardize flow-based assays, participating in regular Proficiency Testing Programs, and collaborating with international consortia to develop and standardize new flow-based technologies.
     
  • Assay Development
    The Analytical Flow Cytometry Component offers assistance in developing novel PFC panels and assays. The Analytical Flow Cytometry Component has developed two methods for designing PFC panels. The first method is a collaborative effort with the Multi-color Flow Cytometry User’s Network (MoFUN) (Reference 1, 2) that involves a through empirical approach to panel development. The second method uses techniques developed by the Analytical Component PFC panel, and is the method is currently used for the majority of PFC panel development.
     
  • Mentoring/Training
    Training is provided for all aspects of flow cytometry from study design through data analysis, and includes key details necessary to build polychromatic panels. The Analytical Flow Cytometry Component provides training for independent operators to use Core cytometers for acquisition. Mentoring/training is provided as a Consultation Service, as Core Operator Assistance, or both depending upon the specific needs of each Investigator.

    In addition, the Analytical Flow Cytometry Component hosts a comprehensive week-long wet-workshop that focuses on “Measuring Antigen Specific T-cells Using Polychromatic Flow Cytometry.” Workshops are offered free of charge to all participants, thanks to the generosity of our sponsors. The goals of the Workshop are two-fold: participants will be able to return to their respective laboratories and perform the advanced 11-color ICS assay illustrated during the Workshop and participants will be able to apply the basic principles reviewed during the Workshop to modify the 11-color ICS assay to suite their own laboratory needs. For more information regarding the next Workshop please visit www.cfar-cig.org.

  • Discounts
    The Analytical Flow Cytometry Component has a strong history of working closely with industry to provide discounted pricing for Core CFAR Investigator’s. We are currently one of six laboratories for participation in the BD Bioscience Key Opinion Leader (KOL) program, which collaborates in the development and testing of new products. In an effort to continue providing Duke CFAR Investigators with the latest technology, the Immunology Core developing similar partnerships with industry. If you are interested in trying some of the latest cutting-edge technology or have an idea for a cytometry-focused collaboration with industry, please contact Dr. David Murdoch.

 

Major Equipment

  • Two optically-matched BD FACSAria cell sorters
    The BSL2/BSL-3 Sorters are operated by a well-trained operators with established procedures approved by Duke’s Biological Safety Division. For more information regarding BSL2/BSL3 sorting services currently available to Duke CFAR Investigators, please contact Dr. M. Anthony (Tony) Moody or refer to the Duke Human Vaccine Institute Shared Resources website.
     
  • One Custom-designed Becton-Dickinson LSRII flow cytometer
    The LSRII analyzer has a four-laser bench (405, 488, 532, and 640nm) and is equipped with 18 fluorescence detectors, in addition to forward and side scatter detectors. Two octagons and two trigons, are coupled with the four lasers, enabling a total of 20-parameters to be measured simultaneously using the LSRII. The LSRII is equipped with a High Throughput Sampler (HTS) to allow input from standard 96-well plates.  (Configuration information and laser layout)
     
  • One Custom-designed Becton-Dickinson LSRFortessa flow cytometer
    The LSRFortessa analyzer has a five-laser bench (405, 488, 532, 635, and 355nm) with a total of 22-parameters. Additional electronics have been added to the LSRFortessa to support two toggle switches for a total of four optical configurations and simultaneous measurement of up to 20 fluorescence parameters. The LSRFortessa optical configuration matches the LSRII for redundancy and backup as needed.  (Configuration information and laser layout)
     
  • One Luminex MAGPIX Multiplexing Instrument
    The MAGPIX is a versatile multiplexing platform capable of performing qualitative and quantitative magnetic bead-based analysis of proteins and nucleic acids in a variety of sample matrices. This system can perform up to 50 tests in a single reaction volume, greatly reducing sample input, reagents and labor. MAGPIX is supported by a broad menu of commercially available assay kits, as well as reagents to build your own assays.
     
  • Two Millipore MUSE Cell Analyzers
    The Muse cell analyzer delivers accurate assessments of cell viability, apoptosis and cell cycle.  It uses miniaturized 3-parameter fluorescence detection and microcapillary technology to perform cell viability, proliferation, and toxicity assays.
     
  • One Advanced Microscopy Group (AMG) EVOS xl Microscope
    The EVOS® xl digital inverted microscope combines advanced ergonomic design, an on-board microprocessor, LED illumination, fully integrated color camera, and user-friendly integrated hardware/software.
     
  • One Shandon Cytospin 2 Cytofuge
    The Shandon Cytospin 2 cytofuge is a low speed centrifuge used to separate and deposit a monolayer of cells on slides while maintaining cellular integrity. Cytofunnels load in a titled position preventing cell loss.  Sealed cover protects user from exposure to aerosoling pathogenic samples. Appropriately stained cytospin slides provide a permanent visual record of cells present in a sample.

 

 

Research Highlights

1. Holl TM, Yang G, Kuraoka M, et al. Enhanced antibody responses to an HIV-1 membrane-proximal external region antigen in mice reconstituted with cultured lymphocytes. Journal of immunology 2014; 192(7): 3269-79. This article demonstrates the use of B cell tetramer reagents to track HIV-1 specific B cells in a mouse model

2. Murdoch DM, Staats J, Weinhold KJ.  OMIP-006: Phenotypic subset analysis of human T regulatory cells (Tregs) via polychromatic flow cytometry.  Cytometry A. 2012 Apr;81(4):281-3. PMID: 22319016. PMCID: PMC3352600.  This article demonstrates the development of a flow cytometry panel for the enumeration of Tregs in HIV infection and other diseases.

3. Murdoch DM, Suchard MS, Venter WD, Mhlangu P, Ottinger JS, Feldman C, Van Rie A, Glencross DK, Stevens WS, Weinhold KJ. Polychromatic immunophenotypic characterization of T cell profiles among HIV-infected patients experiencing immune reconstitution inflammatory syndrome (IRIS). AIDS Res Ther. 2009 Jul 16;6:16. PMID: 19607684.  This article was one of the first of its kind to perform longitudinal T cell phenotyping in HIV-infected patients experiencing IRIS in sub-Saharan Africa.

4. Richards AJ, Staats J, Enzor J, McKinnon K, Frelinger J, Denny TN, Weinhold KJ, Chan C. Setting objective thresholds for rare event detection in flow cytometry. J Immunol Methods. 2014 Jul;409:54-61. PMID: 24727143.  This manuscript demonstrates the flow laboratory’s involvement in the development of novel computational approaches by collaborators in the Duke CFAR Biostatistics & Computational Biology Core.

5. Staats JS, Enzor JH, Sanchez AM, Rountree W, Chan C, Jaimes M, Chan RC, Gaur A, Denny TN, Weinhold KJ. Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays. J Immunol Methods. 2014 Jul;409:44-53. PMID: 24968072.  This article demonstrates the development of PFC panels for the NIH-supported EQAPOL program.

Core Staff Contact

Duke CFAR Immunology Core – Flow Cytometry Component

Surgical Oncology Research Facility (SORF)
915 South LaSalle Street Ext.
Room 119/120
Durham, NC 27710

Leadership:

Dr. David Murdoch
CFAR Immunology Core Co-Director
Flow Cytometry Component Director
david.murdoch@dm.duke.edu
(919) 684-1106

Janet Staats
CFAR  Analytical Flow Cytometry Component Co-Director
jotti@duke.edu
(919) 684-9807

Jennifer Enzor
CFAR Analytical Flow Cytometry Component Lab Manager
jennifer.enzor@duke.edu
(919) 684-3315 (office)
(919) 684-3397 (lab)

Twan Weaver
CFAR Analytical Flow Cytometry Component Specialist
twan.weaver@duke.edu
(919) 684-3315 (office)
(919) 684-3397 (lab)