Analytical flow cytometry is an essential technology for all of the investigators participating in this program project. This flow cytometry core will augment existing capabilities at our institution by providing the first flow cytometer to be housed and operated in our Biosafety Level 3 laboratories. Funds for the purchase of this instrument have already been committed by the institution, and we anticipate the purchase and installation of a Becton-Dickinson FACSCalibur analyzer with low speed sorting capability before the initiation of this program grant. This instrument will allow the performance of up to four color fluoresence single cell analysis of cell suspensions harvested from mice infected with virulent or potentially virulent strains of M. tuberculosis. Multicolor FACS analyses using a range of monoclonal antibodies to identify and quantitate specific cell populations will be carried out routinely by this facility, as will staining of specific T cell populations using peptide loaded MHC class I tetramer reagents. Analysis of apoptosis, cell cycle progression and calcium flux will also be available in a BSL-3 environment through the establishment of this core. Limited cell sorting capabilities that should be adequate for the proposed isolation of serologically modified mutants of M. tuberculosis will also by possible with this core facility. The core director, Dr. Steven Porcelli, was first trained in the use of analytical flow cytometers in 1989, and has been actively using these instruments in his own research continuously since that time. He also is currently the director of the flow cytometry facility of the NIH funded AECOM Center for Aids Research, which provides a BSL-2 environment for analytical flow cytometry as well as limited sorting capability using a Becton-Dickinson FACSort instrument. He is experienced in training and supervisingtechnicians and other laboratory personnel to carry out flow cytometry procedures. In establishing this core facility, he will recruit and train an appropriate individual to serve as a dedicated flow cytometry technician to carry out the bulk of the day to day operation of the core.
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