Analysis and separation of murine bone marrow stem cells by H33342 fluorescence-activated cell sorting

Murine bone marrow cells were stained with the fluorescent bisbenzimide dye H33342, a supravital DNA stain, and sorted on the basis of differences in fluorescence intensity by a light-activated cell sorter. Sorted cells were submitted to assays to enumerate spleen colony forming cells (CFUs), HPP-CFC and GM-CFU-1 and -2. The recoveries of these cell types after the separation was 70 to 120%, except for GM-CFU-2 (10-40%). The frequency distribution of these cell types with respect to their fluorescence intensity suggested that the majority of CFUs and HPP-CFC are quiescent, whereas GM-CFU-2 are proliferating. Furthermore, HPP-CFC could be separated from GM-CFU-1 and -2 on the basis of fluorescence intensity differences, which indicates that these cell types are different. CFUs determined 10 days after irradiation and grafting of the recipient mice were distributed evenly over the two fluorescence intensity subpopulations which contained most of the HPP-CFC and the GM-CFU. The same was observed at day 8. However, CFUs determined at day 12 were only present in the subpopulation of low fluorescence intensity which also contained most of the HPP-CFC. This observation provides evidence for heterogeneity of the spleen colony forming cells.