Flow Cytometry

University Of Tx Md Anderson Can Ctr, Houston TX

Investigators

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Abstract

The Flow Cytometry and Cellular Imaging Core Facility (FCCICF) provides cellular analysis to investigators with peer-reviewed grants. The FCCICF has two sites, on the north campus and on the recently-opened south campus. It occupies 1900 sq. ft. and is directed by Dr. Michael Andreeff. The FCCICF develops and provides techniques for single-cell analysis. Cell phenotyping, proliferation, signaling and apoptosis assays have been established and modified for multiparametric analysis. Immunophenotypic analysis was combined with assays of intracellular proteins related to apoptosis (Bcl-2, Bcl-XL, BAG-1, p53, Rb, caspase activation, mitochondria! membrane potential and Fas), cell signaling (MARK), proliferation (Ki67, cyclins, BrDU, PCNA, DNA) and cell division history (CFSE). Quantitation of cellular antigens allows determination of the antibodybinding capacity per cell. Rare events and progenitor/stem cell subpopulations can be detected and isolated by three-laser excitation/eight-parameter fluorescence-activated cell sorting (FACS) for subsequent analysis by molecular cytogenetic and other molecular techniques including quantitation of intracellular PKCoc, Bax, Bcl-2, ERK, pERK, XIAP by laser scanning cytometry. FISH has been combined with apoptosis assays to discriminate apoptosis in normal and malignant cells. The number, phenotype and proliferation of minimal residual disease cells can be determined at levels of one malignant in 30,000 normal cells. Methods for detection of transgene expression in cells are in place using p-galactosidase (p-gal), nerve growth factor receptor (NGF-R), and green fluorescent protein (EGFP). Acquisition of 3 new FACS Aria cell sorters and a four-laser flow cytometer (B&D LSRII) has upgraded the facility to provide state-of-the-art isolation and analysis. Laser confocal microscopy has been used extensively and was upgraded by acquisition of an Olympus FV-500 multi-user instrument and a DSU spinning disc confocal system. High-impact studies in cancer prevention, growth factor signaling in breast cancer, apoptosis regulation in leukemias and multistep tumorigenesis all utilized confocal microscopy. The FCCICF now utilizes 17 major instrument systems. The Core supports numerous R01, P01, R21, and SPORE grants. Since the previous review, the number of users has increased 145% (169 investigators with peer-reviewed grants from 19 different programs). 67% of users have peer-reviewed funding. During the previous funding period, 11,668 hours of service was provided. Use has tripled to greater than 7,000 hours estimated for 2007. Future plans are focused on the continued development and application of cutting-edge technologies in cell analysis.