This proposal is for the establishment of a Flow Cytometry Core Facility (FCCF) within the proposed Center for AIDS Research at the University of Alabama at Birmingham. The establishment of the CFAR FCCF will provide access to all investigators performing AIDS- related research using potentially infectious material to a state of the art, computer controlled four parameter (2 channels of fluorescence plus forward and orthagonal light scatter) flow cytometer/sorter. This unit will be equipped with a Coherent Innova 2 watt argon-ion laser, which when operating at 488 nm will allow the discrimination of lymphocytes, monocytes, granulocytes, erythrocytes and dead cells using bivariate light scattering analysis. In addition, this laser line will excite both fluorescein isothiocyanate (FITC) and phycoerythrin (PE), and one or two color immunofluorescence analyses will be possible. Cell cycle analyses can also be performed using this cytometer using 488 nm light. Acridine orange (AO) staining of permeabilized cells will be used to independently quantitate the DNA and RNA present in individual cells. Propidium iodide (PI) staining is an additional method for quantitating cellular DNA, and offers the advantage that it can be combined with FITC immunofluorescence to yield data on cycle-dependent antigen expression. These types of experiments are not currently possible on HIV-infected material at UAB.
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