In this revised Shared Instrumentation Grant application, funding is requested for a FACS VantageTM Flow Cytometer (FACS) to be located within the biosafety level 3 (BL-3) containment laboratory of the Gladstone Institutes of Virology and Immunology (AIDS Research Center) at the San Francisco General Hospital (SFGH) and University of California, San Francisco (UCSF). This new Institute represents one of the largest basic science centers in the world dedicated to the study of HIV and AIDS. The proposed FACS will be used for fluorescence based assays and purification of live cells infected with HIV- l. Given the biohazards of such work, the FACS will be specifically modified with a special biohazard containment system that safely reduces the hazardous aerosols generated by "stream in air" cell sorting. In addition, the FACS will be housed within the certified BL-3 containment laboratory of the Gladstone Institute. At present, no other cell sorters in San Francisco can be used to separate have HIV- l infected cells due to the lack of appropriate biosafety equipment and access to a BL-3 containment laboratory. As such, this new instrument would significantly strengthen and enhance the research capacity of many NIH-funded Gladstone, SFGH and UCSF scientists engaged in AIDS research. The scientific interests and expertise of the selected major users cover a wide spectrum of HIV biology; consequently the FACS will critically support a broad range of projects including l) molecular analysis of the pathogenic effects of the. HIV- l nef gene, 2) biochemical studies of the HIV- l tat gene and the identification of specific tat antagonists, 3) characterization of a "second" receptor for HIV- l involved in gp4l mediated cell fusion and viral entry, 4) molecular analysis of AIDS-related lymphomas, 5) dissection of the potential pathogenic role of deranged cytokine production and antibody-dependent cellular cytotoxicity in pediatric AIDS, and 6) delineation of the molecular determinants involved in maternal-fetal transmission of HIV-l. In summary, this proposed FACS is pivotal to the success of this new AIDS Research Center and will provide a critical resource to the entire Gladstone/UCSF/SFGH academic community involved in AIDS research.