CORE B - FACS/Immunoassay
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The objectives of this Core are: (1) to provide acessible and cost saving access for DDC investigators to flow cytometry, cell sorting, and immune monitoring services; (2) to facilitate DDC user access to innovative cell and immune monitoring technologies; (3) to encourage digestive disease-focused research by investigators in diverse disciplines through subsidized support for faciliy usage.
The goal of the FACS component is to provide DDC investigators with access to state-of-the-art flow cytometry at a favorable cost and with the easy access required for the efficient pursuit of immunologic and cell biologic investigations. At the VA, the FACS Core is run by the DDC itself, while at Stanford, the DDC subsidizes the use of the Stanford Shared FACS Core facility by DDC RBIs. Most investigators have traditionally faced barriers to the use of flow cytometry and cell sorting resulting from lack of experience, and/or the very high equipment cost, or the high cost of non-subsidized core services. Our Core is designed to overcome these barriers for DDC investigators by providing easy and relatively inexpensive access to equipment in conjunction with a support system for assistance and training. Although full assistance at the VA is available during normal working hours, experienced users can also use the facility during nights and weekends. For the FACS component of this Core, users are not charged for their utilization. Ms. Rott tracks Core utilization with the assistance of the DDC Administrator. Reservation of time is either by sign-up book on site or by contacting Lusijah Rott by phone or email.
The Human Immune Monitoring component will leverage another Stanford Service Center, the Human Immune Monitoring Center (HIMC), to provide full-service Luminex assays (for 26 mouse or 50 human cytokines and chemokines) and CellBiosciences Nanopro "Firefly" automated capillary isoelectric focusing assays (for phosphoprotein analysis of cell lysates). These are two of the standard assay services offered by the HIMC that have been of particular interest to DDC investigators. The HIMC has also purchased a CyTOF for high dimensional research (e.g. simultaneous detection of 30 or more antigens oer cell). An important goal of Core B is to help deveop key reagents (isotopicially labeled MAbs) and services (training and assistance in isotope labeling) to DDC members, and to integrate the revolutionary CyTOF system into our Flow Cytometry offerings. For luminex and other HIMC services, the DDC will subsidize up to $500 per RBI per year.
Functions and Activities of the Flow Cytometry Component
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Assistance and training in appropriate experimental design for flow cytometry and fluorescence-activated cell sorting experiments.
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Training and special immunofluorescence staining procedures for flow cytometry, including, but not limited to studies for up to 14 colors, apoptosis, DNA cell cycle analysis, bead assays, and studies examining kinetic changes.
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Fully assisted bulk and single cell sorting - FACS ARIA II and FACS Aria III.
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Fully assisted analysis - FACScan, FACScaliber, LSRII, Fortessa.
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Facility maintenance and troubleshooting.
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Training in flow cytometry applications and in individual use of the benchtop cytometers. Training for independent sorting on the two Arias.
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Independent, unassisted use of the bechtop cytometers and sorters by trained researchers during regular hours and on evenings and weekends.
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Subsidized access to FACS data analysis software (FlowJo) through a Site License. The Shared facility maintains a site license for use of FlowJo data analysis software. Facility users obtain FlowJo at a discounted rate of $195 per computer per year. DDC investigators using the Shared facility will have the option of using a DDC subsidy of 50% of the license fee to participate in the site license, and to download FlowJo onto their own computers, significantly facilitating analysis.
Functions and Activities of the Immunoassay Component
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Discounted and professionally run Luminex Bead Arrays, which provide the means to simultaneously assay up to 26 mouse or 51 different human cytokines in 100 ul samples.
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The NanoPro System for Signaling Analysis of Tissue and Cell Samples (Cell Biosciences) is an automated, rapid and quantitative protein measurement apparatus that combines nanofluidic isoelectric focusing with antibody detection to measure specific protein amounts and degrees of post translational modification, such as phosphorylation.
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The new CyTOF (for Cytometry by Time Of Flight mass spectrometry) flow cytometry system, allows simultaneous analysis of 30 or more analytes per cell. CyTOF mass spectrometry is a highly innovative multiparameter technology to study cell subsets and cellular responses.
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Other standardized assays performed by the HIMC include standardized flow cytometry immunophenotyping and phosphoepitope staining, gene expression and miRNA arrays.
The DDC FACS Lab, with post doctoral fellow Guangin Li at the LSRII
