The mission of the Immunology Core is to further innovative, interdisciplinary and translational research that enhances our understanding of the pathogenesis and immunopathogenesis of HIV/AIDS; provides new approaches toward understanding cellular, humoral and innate responses to HIV; and develops novel HIV therapy and vaccine strategies. To achieve these goals, the Core provides state-of-the-art immunological services and reagents; specialized technology; leadership, expertise and advice; and collaborative support in the area of immunological research to the Penn CFAR community. The Core also works closely with the Perelman School of Medicine Human Immunology Core (HIC).
Immunology Core Services
The Immunology Core provides a wide range of services and reagents to benefit the immunological research of the Penn CFAR Community. Services are provided by facilities in both the University of Pennsylvania and the Children's Hospital of Philadelphia Research Institute and are housed at several sites. Services include:
- Primary Cell Purification (Riley Lab, 8th Floor Smilow Center for Translational Research)
- Freshly isolated primary PBMCs, CD4, CD8, T cells, NK, monocytes and B cells
- CCR5 deficient T cells and monocytes from Δ32 donors
- Standard Immunological Assays (12th Floor Abramson Research Center, CHOP)
- Cell Based Assays
- Flow Based Assays and Services
- Cellular, Humoral, and Molecular Immunology Assays (HIC, 410 & 704 Stellar Chance)
- Specimen Processing, Banking, & Sample Shipment
- Luminex assays
- Flow Cytometry
- Digital ELISA (Simoa)
- Ultra-sensitivity- 1,000-fold improvement over today’s state-of-the-art immunoassays
- Measure proteins in single cells
- Precision- Exceptional repeatability at ultra-low concentrations of target proteins
- Full Automation- Enhanced data reproducibility
- Wide Dynamic Range- Proprietary combination of both digital and analog signal measurements provide > 4 logs of dynamic range.
- Multiplexing Capability- Currently up to 4 different analytes can be tested from a single sample, future capabilities of 10 plus analytes
- Measure neurology markers in serum/plasma/exosomes, not just CSF
- Custom assay development offerings (develop your own specific assay)
- Measure markers in a wide variety of sample matrices (Serum, Plasma, CSF)
- PCR levels of sensitivity of detection of proteins (such as p24)
- Detect “normal” endogenous levels of proteins, unmeasurable with conventional technologies
- Over 60 off the shelf kits available today or develop your own homebrew assays with ease
- Sequencing of Human & Murine IgH rearrangement
- Sequencing of Human TCR Vβ rearrangements
- Amplification and cloning of Ig rearrangements
- Data analysis pipeline for repertoire studies
- Customized Data Analysis
- Scientific Consultation and Assay Development
- BSL-3 Cell Sorting Facility
- Small Animal Model of HIV-1 Infection
Primary Cell Purification
Within the last several years there has been a greatly increased demand for primary cells in both HIV and immunological research. There are multiple reasons for this. There has been a shift in the field from the use of cell lines to primary cells for research. For example, during the transition from primary cell to transformed cell line, critical signal transduction pathways are altered, which complicates the extrapolation of data to primary cells. Moreover, certain cell types, such as dendritic cells (DC), do not have cell lines that faithfully model their activities, necessitating the use of primary cells. Lastly, certain experiments require cells with a particular genotype or HIV status. Transformed cell lines are popular because they are inexpensive, require little expertise to use, and are easy to propagate; the major goal of this Immunology Core service is to make primary cells as accessible and easy to use as transformed cells.
Dr. Riley and his lab have been purifying primary human cells for research since 1995 and distributing to others as a function of the CFAR Immunology Core since 2003. For the most part, investigators prefer cells that have been purified via a negative selection process, which leaves them as minimally manipulated as possible. Cells are obtained from healthy donors by leukapharesis, and depending on users' need, are separated into subsets by negative selection using Rosette-Sep platform developed by STEMCELL Technologies. Cells are generally provided fresh to investigators or, for those who require large batches of single-donor cells to be used at different time points, can be provided as frozen aliquots. The cell purity is always greater than 90% and is routinely greater than 95%. During the time Core E has served the Penn CFAR community, we have isolated cells from greater than 600 apheresis products; a sample summary for one such product is shown in Table 1. Several investigators have requested purified cells from HIV-1 infected donors. This can be set up on a case by case basis. These cells are only available to investigators who have specifically requested cells from HIV-1 infected donors.
Table 1. Typical Cell Request
|Cell Type||# Requested||Purity|
|CD4 T cell||350 million||95%|
|CD8 T cell||100 million||97%|
|CD8-depleted PMC||100 million||98%|
This is an ideal core function because the effort required to isolate 10 million cells is the same as that to isolate one billion cells. Rather than having all laboratories who wish to work with primary cells obtain their own IRB-approved protocol, recruit donors, and develop the expertise to purify cells, the Immunology Core performs all of these functions, and since the entire apheresis product is utilized, the price per cell is much less than if the researchers were to obtain their own cells. A major part of the appeal and popularity of this service is that it removes the regulatory paperwork required to work with primary cells: The Immunology Core maintains the IRB-approved protocol and the individual core users receipt of these cells is considered secondary use of de-identified human specimens which is not considered human use by both NIH and our IRB. This recent clarification of what human use is removes a significant burden to use primary human cells to study HIV-1. We have found that the decreased regulatory burden often makes it more attractive for labs to carry out even pilot experiments with primary human cells, and not resort to cell lines. We do maintain an inventory of frozen cells so please inquire if you would prefer frozen cells.
Donors are not tested for any infectious agents. So as with all blood borne products, users are encouraged to follow universal precautions.
Order cells at the Human Immunology Core
Primary Contact: James L. Riley, Ph.D. | email@example.com | 215-573-6792
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Standard Immunological Assays
Cell Based Assays
Contact Rich Tustin at firstname.lastname@example.org or 215-590-2043 for pricing and scheduling.
- Functional Natural Killer Cell Assay (FNKA)--The lab is CLIA certified for this assay, so the results of the assay can be used for patient management. The functional capability of NK cells is determined in vitro by exposing them to chromium labeled NK sensitive target cells. NK cell activity is quantified in this assay by the measurement of chromium released from NK cell lysed targets. This lytic activity is normalized to reflect the number of peripheral blood mononuclear cells and natural killer cells per 107 cells required to lyse 20% of the chromium-labeled targets. This is referred to as the LU20 PBMC and LU20 NK, respectively.
- Cytokine assays on cell culture supernatants and biological fluids by, Multiplex (Luminex), ELISA or ELLA. Pricing is calculated as the cost of the kit plus labor.
- Other assays and services are available upon request (separation of PBMC from whole blood, separation and cryopreservation of plasma, serum, etc)
Flow Based Assays and Services
Contact Florin Tuluc at email@example.com or 267-426-5350 for pricing and scheduling.
Sample processing (surface and intracellular staining), functional assays, analyses, and access to flow cytometry analyzers are offered on a fee-for-service basis. Most common assays include immunophenotyping of human blood samples from clinical studies:
- Flow cytometric, single platform CD4 counts (CLIA and IQA certified)
- Custom multi-color immunophenotyping of leukocyte subpopulations :
- leukocyte subsets (T, B, NK cells, monocytes)
- T cells subsets (naive, memory)
- immunoregulatory cells (Tregs)
- NK cells: expression of inhibitory/activating NK receptors
- Circulating stem cells and progenitors
- Intracellular cytokine panels
- Custom panels of antibodies
The laboratory routinely performs full service testing on whole blood as well as fresh and cryopreserved PBMCs. Custom panels of antibodies are available upon request.
Functional flow cytometry and bead-based assays are also available as full-service to our users:
- Cell proliferation and viability
- Cell cycle
- Mitochondria membrane potential and generation of reactive oxygen species; mitochondrial mass
- Intracellular calcium assays by flow cytometry
- Protein phosphorylation using phospho-specific antibodies
- Cytometric Bead Array for TH1/TH2 Cytokines
- Cytometric Bead Array Flex Human or Mouse custom cytokine assays
- Other flow cytometry assays are available upon request.
Four analyzers are available in our facility and they may be operated by trained users:
- LSRFortessa (blue, red, yellow-green, violet and UV lasers; 18 color detectors).
- CytoFLEX LX (blue, red, yellow-green, violet, UV, and infrared lasers; 21 color detectors)
- CytoFLEX S (blue, red, yellow-green, violet; 13 color detectors)
- LSRII (blue, red, and UV lasers; 8 color detectors)
- FACSCalibur (blue and red lasers; 4 color detectors)
- Accuri C6 (blue and red lasers; 4 color detectors)
Cell sorting services are performed on a daily basis on four sorters:
- FACSAria Fusion (blue, red, yellow-green, violet; 15 color detectors)
- MoFlo Astrios EQ (blue, red, yellow-green, violet; 13 color detectors)
- FACSJazz (blue, red, yellow-green lasers, 6 color detectors)
- Biosorter (Union Biometrica; blue and yellow-green lasers, three color detectors, 250 and 1,000 um FOCAs)
All sorters may be operated by trained users.
Cell-Based Assays: Rich Tustin | firstname.lastname@example.org | 215-590-2043
Flow Cytometry Assays and Services: Florin Tuluc at email@example.com | 267-426-5350
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BSL-3 Cell Sorting Facility
The BSL3 Cell Sorting Facility allows investigators working with pathogenic agents such as HIV, SIV, Hepatitis and HTLV-1, among others, to carry out flow cytometry and cell sorting with live, unfixed material. By having access to a state-of-the-art cell sorter, users of the facility are able to perform most sophisticated flow cytometric analyses.The facility is equipped with a BD FACSVantage SE flow cytometer from BD Biosciences (San Jose, CA) with a wide array of options. The TurboSort Plus™ option allows sorting at higher sheath pressures (up to 60psi) and generation of higher drop drive frequencies (up to 99K), permitting high-speed sorting. The BD FACS DiVa Option takes advantage of the latest digital electronics, permitting faster signal processing, full interbeam compensation, four-way sorting (QuadraSort™), software compensation (via a matrix spillover coefficient) and new sort masks for improved conflict resolution (resulting in better purity and sort yields). The sorter is also equipped with the BD Aerosol Management Option to rapidly evacuate aerosolized particles within the enclosed sort chamber. The unit has 3 lasers and 8 fluorescence detectors.Along with 2 detectors for light scatter and time, 11 parameters are available, with future additions permitted by the digital electronics. The primary laser is a four-watt water - cooled argon laser, which can be easily tuned to 488nm or 514nm excitation wavelengths. The second laser is a six-watt water-cooled krypton laser, which can provide excitation in the violet (407nm or 413nm) or ultraviolet (351 and 356nm). The third laser is an air-cooled red helium-neon laser emitting at 633nm. This combination provides a wide array of useful excitation laser lines, from ultraviolet to near-ultraviolet and throughout the visible spectrum.
The Core Facility is equipped and experienced to meet the needs of our investigators for applications such as high-speed sorting, eight color sorting, immunophenotyping cell tracking, cell proliferation, apoptosis, cell viability, DNA Total Content, SP cells (Hoechst 33342), fluorescent proteins (CFP, GFP, YFP, and RFP), FRET studies (CFP/YFP) and calcium studies (Indo-1). Auto Cloning into 96-well plates and four-way sorting is also readily available. Additional protection is afforded to the operator, wearing a disposable gown and hood, and breathing HEPA purified air through a powered air-purifying respirator (PAPR). The cell sorter is located in a BSL3 suite, with controlled access limited to the operator
Visit the Flow Cytometry & Cell Sorting Resource Laboratory website for more information including pricing, contacts, and scheduling resources.
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Small Animal Model of HIV-1 Infection
The Penn CFAR Immunology Core is partnered with the Xenograft and Stem Cell Core to provide "ready-to-use" NSG and BLT mice, experimental planning and design, IACUC protocols, and in vivo experimental techniques.
Robust human T cell engraftment was developed in 2005 using mice engineered to have common gamma chain deficiency, the Nod, SCID, common gamma chain deficient (NSG) mouse. This model has been used to study approaches like ZFN targeting coreceptors and anti-sense RNA to protect fully mature CD4 T cells from HIV-1 infection in vivo. As an important priority for the coming cycle, the Penn CFAR Immunology Core has been working with Xenograft and Stem Cell Core to establish the next generation humanized mouse model, the Bone marrow-Liver-thymus (BLT) humanized mouse.
With support from the CFAR, the Xenograft Core monitors engraftment to determine when the mice are ready to use, at which point the mice are transferred to the investigator's protocol. In addition to producing "ready-to-use" BLT and NSG mice, the Core can further support users by performing a wide variety of in vivo techniques including injections of live HIV virus, human T-cells, peripheral blood sampling and tissue collection. The Core Director is also able to assist CFAR investigators with experimental planning, experimental design, and IACUC protocols.
Primary Contact: James L. Riley, Ph.D. | firstname.lastname@example.org | 215-573-6792 for more information regarding the small animal model service.
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