The Flow Cytometry Core (FCC) will provide three primary services to support the scientific aims of the four proposed projects;population purification using cell sorting, cytokine identification and quantification using bead based cytokine arrays (Luminex system), and multispectral imaging flow cytometry (MIFC) using the ImageStream system. The first of these services, cell sorting, will be accomplished using a BD DiVa FACSVantage high speed sorter or the recently acquired i-Cyt Reflection high speed sorter. Both of these instruments are full equipped to sort the populations of interest described in the aims of Projects 1, 2, and 3. Evaluation of cytokine profiles of serum/plasma, tissue homogenates and culture supernatants will be accomplished! using the Luminex bead based multiplexing technology. Screening of up to 32 analytes will be available as well as customized panels of analytes of specific interest to accommodate the aims of all four projects. The FCC will provide sample preparation, data acquisition and data analysis for these assays. Thirdly, with the use of the ImageStream imaging flow cytometer, multispectral images as well as statistically robust data will be available to evaluate and confirm protein colocalizations, cytokine production, and cellular activation, as well as provide additional morphological information on cellular populations from the lung, spleen, liver and lymph node. In addition, the FCC has available instrumentation should the need arise to support polychromatic flow cytometry in excess of the capability of the Project leaders'own instrumentation. The FCC will also provide consultative services to the Project leaders in terms of fluorochrome selection, sample preparations, data analysis, and data interpretation. Likewise, the FCC Core Director will coordinate with the Director of the Histology Core (Core B) to insure comprehensive and complimentary services for all four Projects for the evaluation of immune interactions in the process of viral clearance and tissue injury. Flow Cytometry Core will provide the tools to a)isolate specific subsets of cells to further investigate their role in this process, b) identify and quantify the milieu of secreted cytokines/chemokines driving the response, c) capture morphological and fluorescent images of specific cell types identified as key components.
Availability of the appropriate Flow Cytometric technologies is critical to the success of the proposed projects which attempt to dissect the role of the host's innate and adaptive immune responses in viral clearance and tissue destruction from inflammatory responses.
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