Flow Cytometry Techniques in Radiation Biology

Considerable evidence exists that all blood cells are derived from HSC. These cells are of interest to radiobiologists because they are highly sensitive to low doses of ionizing radiation. Hematopoietic stem cells HSC are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC. Because of the importance of HSC in the postirradiation syndrome, we have developed a new rapid method based on flow cytometry not only to assay but also to purify and characterize HSC. This new method makes extensive use of non-clonal antibodies conjugated to fluorescent phycobiliproteins through the sulfhydryls of the hinge region of the IgG molecule. An optical bench arrangement with a dye laser and an argon laser was used to dual excitation of the phycobiliprotein-monoclonal antibody conjugates and various cellular and DNA probes. Using 4, 6-diamidino 2-phenylindole dihydrochloride DAP exclusion to identify viable cells, it was possible to follow regeneration of postirradiated rat marrow HSC. Reprints.