Fluorescence-Activated Cell Sorting (FACS) and Molecular Cytogenetics (FISH)

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The FACS/FISH/Confocal Microscopy Core provides functional, phenotypic, cell cycle, genotypic and structural analysis to the investigators of this program project. The laboratory has developed cutting edge techniques in single cell analysis. For the quantitation of apoptotic cells, the TdT-b-dUTP assay (TUNEL) was modified for multiparameter analysis in combination with DMA and BUdR measurements (cell cycle, ploidy). Also, phenotypic analysis of stem and progenitor cells (Project 1), analysis of intracellular proteins related to apoptosis (p53, MDM2, bcl-2, bax, XIAP) (Project 1), and of cell surface antigens including fas and MDR1 is provided. Expression of new regulators of apoptosis proteins (Projects 1, 2) will be analyzed in normal and leukemic cells. Binding of Annexin V to phosphatidyl serine (PS) is utilized for testing changes in the membrane lipid structure associated with apoptosis. A newly developed assay identifies apoptotic cells (PS/Annexin V+) in the AML stem cell compartment (CD34+38"123*). New assays to measure changes in the mitochondrial membrane potential and of cytochrome c in cells initiating apoptosis (CMXRos) and the detection of cleaved caspase 3 are conducted for Projects 1,2,3 and 4. Quantitation of cellular antigens allows us to determine the Antibody Binding Capacity (ABC). CD34 cells are MACS separated for subsequent analysis by FACS. Cell kinetic changes are determined by DNA/Ki67/ CD34/CD38 FCM and the number of actual cell divisions is determined by PKH26 labeling, with cells undergoing none or up to ten divisions being separated by FACS for subsequent molecular analysis (Project 1). Fluorescence in situ hybridization (FISH) determines the number of clonal leukemic interphase or metaphase cells (Projects 4, 5) before and after treatment with the CXCR4 inhibitor AMD3100. The combination of FISH and TUNEL or PS/Annexin V assays allows us to discriminate apoptosis in normal and leukemic cells (Projects 1 and 3) in mixed cell populations. Finally, progenitor and stem cell compartments (CD34+38~, CD34 38~123+ and CD34" lin" cells that eliminate Hoechst 33342, so-called "SP" cells) are sorted for determination of mRNA and protein levels by RPPA. Projects 1 and 2, are analyzed for the presence of clonal leukemic and normal cells. Many of the clinical trial translational studies (BH3 mimetic GX015-070MS, XIAP antisense AEG35156, Raf inhibitor BAY 43-9006 and CXCR4 inhibitor AMD3100 in Projects 4 and 5 are being conducted in Core B1. In addition to multiparametric flow cytometry and cell sorting, the Core provides Laser Scanning Cytometry for quantitation of antigen/proteins in single cells. Importantly, laser confocal microscopy has become an indispensable tool for analysis of intracellular localizations of many proteins, e.g. p53 and AIF (Projects 1,2). The Core provides critical support for all Projects.