This grant application requests NIH support for the purchase of a BD Biosciences LSR II Blue Laser flow cytometer with four lasers. This very sophisticated piece of equipment will allow our user group to take advantage of the fastest developments in fluorescent technology. Our group of applicants (four major users and three minor users) all work on infectious immunology and rely heavily on cytofluorimetry as a major experimental technology. The LSR II is a highly sensitive and versatile cytometer which will vastly improve the efficiency with which we can measure expression of cell surface and intracellular proteins, assay for in vivo cytotoxicity, and perform DNA cell cycle analysis. This instrument is not currently available on campus for use with BL2/BL3 samples. Use of this instrument will make it possible to detect very rare populations from small sample sizes. Members of our group study very minute cell populations such as brain-infiltrating lymphocytes, memory CDS T cells in nonlymphoid tissue, specialized macrophage subsets, and pathogen-induced single granuloma lesions. In addition, this instrument would allow us to use new fluorescence markers that cannot be detected by our current cytofluorimeters. These new markers are important because they will allow the use of new multicolor transgenic or retrovirus-mediated in vivo labeling of cells and multi-color infectious agents. Finally, all of us are very experienced in 4-color flow cytometry; however, our projects would benefit greatly from being able to measure more parameters from the same samples. The analysis of 10 to 14 parameters in an interdependent way is not currently possible using 4-color cytometry, and is particularly important for both the limiting sample sizes discussed above and samples of experimental rarity, such as lymph nodes or mucosal samples prepared from SIV infected rhesus macaques where we want the most possible information from the least possible number of animals. Analysis of unfixed samples from animals infected with diverse pathogens including Histoplasma and Trypanosoma would be a real advance to our research programs. Advances in fluorescence technology have exploded in the past few years, especially as applied to infectious immunology. A new generation of flow cytometric assays is becoming available and our group of users would like to advance to the next level. ? ?
