We find tht normal hypaque-ficoll freshly isolated (unculturd) PBl from heparinized whole blood show some degree of apoptois. Afer fixation with 0.3% paraformaldehyde and 70%EtOH, the cells are treated with Tdt and BrdUTP at 37 degrees C for 1 hr. The control for these experiments include the treatment of cells identically but without the addition of Tdt. Cells are stained with FITC anti-Brdu monoclonal antibody and propidium iodine (PI) according to the Pharingen kit. A FACScan flow cytometer with a doublet discrimination modlule (DDM0 and CellQuest software are used. In a series of three experiments, the estimated percentage of positive cells ranged from 3-19%. Inspection of the two parameter histograms suggest non-specific binding. Have others seen this? Is there a way to reduce it? It makes it difficult to measure low levels of spontaneous proliferation. Comments will be appreciated. Gerald E. Marti, MD, PhD, Chief Flow and Image Cytometry Section Laboratory of Medical and Molecular Genetics Division of Cell and Gene Therapies CBER FDA NIH Bdg 29B Rm 2NN08 8800 Rockville Pike Bethesda, MD 20892 301-827-0453 FAX #301-827-0449 Email: gemarti@helix.nih.gov
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