The cell analysis and FACS Core facility provides genotypic and phenotypic analysis and separated cell populations to the investigators of this program. The laboratory has provided and developed cutting edge techniques in single cell analysis. For the quantitation of apoptotic cells, the Tdt- b-dUTP assay (TUNEL) was established and modified for multi-parameter analysis in combination with DNA and BUdR measurements (cell cycle ploidy). Also, immunophenotypic analysis of progenitor cells and lineage restricted cell populations, analysis of intracellular proteins related to apoptosis (bcl-2, bax, p53), and of cell surface antigens including fas and MDR1 is provided. Recently, binding of Annexin V to phosphatidyl serine (PS) was recognized as a test for changes in the membrane lipid structure that precedes changes detected by TUNEL in cells initiating apoptosis. Quantitation of cellular antigens allows us to determine the Antibody Binding Capacity (ABC). Retrovirally-transduced cells will be analyzed for expression of transgene (NGF receptor) and FACS-sorted CD34 cells will be separated for subsequent analysis by MACS, and CD34 subpopulations by MACS/FACS. Cell kinetic changes can be studied in vitro and the number of actual cell divisions can be determined using PKH26, with cells undergoing none or up to ten divisions being separated for subsequent molecular analysis. Fluorescence in situ hybridization (FISH) to determine the number of t(9,22) containing interphase cells has been established. The combination of FISH and TUNEL or PS/Annexin V assays allows us to discriminate apoptosis in normal and leukemic cells. A novel assay has been developed to determine bcr-abl transcripts in situ by RT- PCR. This assay will be compared to hypermetaphase FISH and competitive RT-PCR for the detection of low levels of residual leukemic cells. Finally, progenitor cell compartments (CD34 and """"""""SP"""""""" cells) will be analyzed for the presence of Ph cells, based on our observation that normal but not CML cells express MDR1 and that fas (CD95) is expressed more in CML than in normal progenitor cells.
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