The Fluorescence Microscopy Core will be housed at NYU School of Medicine and will provide PPG members access to three key fluorescence microscopy technologies for studies on PD-1 expression and function. The key biological questions relate to the impact of PD-1 on HIV specific T cell degranulation (Projects 1 and 3), the expression pattern of PD-1 message in antigen specific T cells (Project 2), the impact of PD-1 on T cell sensitivity to antigen (Projects 3), and the effect of PD-1 on organization of signaling molecules in the immunological synapse (Projects 3 and 4). The core will focus on providing both the assay infrastructure (in conjunction with Core B) and instrumentation necessary to address these questions. The assay technologies will include biochemical infrastructure required to analyze degranulation and signaling in immunological synapses, to count peptides in immunological synapses, and analyze single locus gene expression patterns by RNA-tluorescence in situ hybridization (RNA-FISH). Reagents for both mouse (Project 2 and 3) and human (All Projects) systems will be established. The available instrumentation includes a microscope equipped for total internal reflection fluorescence microscopy (TIRFM), wide-field fluorescence microscopes calibrated for 3D counting of phycoerythrin tagged antigenic peptides and fluorescence resonance energy transfer experiments, and confocal microscopes optimized for 3D localization of discrete fluorescent signals in the nucleus and quantitative co-localization studies in cell-cell synapses. Funds toward installation of a second TIRFM system are requested to increase capacity for analysis of rare antigen specific cell populations (Projects 1 and 4), which require longer imaging sessions to acquire data. Michael Dustin (Principle investigator and Core Director) will direct the core and coordinate degranulation, signaling and FRET studies (Projects 1, 3 and 4). Michelle Krogsgaard (Co-investigator) will oversee single molecule counting experiments (Project 3) and Jane Skok (Co-investigator) will oversee RNA-FISH experiments (Project 2) all performed by a dedicated technician.
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