Separation and functional analysis of bone marrow cells separated by rhodamine-123 fluorescence

Mouse bone marrow (BM) cells were separated on the basis of fluorescence intensity after labeling with the supravital, fluorescing dye rhodamine 123 (Rh 123). Rh 123 accumulates in the mitochondria. The BM fractions were tested for the presence of spleen colony-forming units (CFU-S) that produced colonies at day 8 and day 12 after bone marrow transplantation; for the ability to rescue lethally irradiated mice; and for thymus-repopulating ability. The results showed that all the day-8 CFU-S incorporated a relatively large amount of Rh 123, while the day-12 CFU-S were stained heterogeneously. Survival after lethal irradiation, as expressed in the numbers of day-12 CFU-S transplanted, was predominately mediated by the weakly fluorescing fraction. However, early thymus repopulation, which is caused by prothymocytes contained in the graft, was mediated by the brightly fluorescing fraction. Data in the literature indicate that a high uptake of RH123 is correlated with cellular proliferation. This suggests that all the day-8 CFU-S and about 60% of the day-12 CFU-S are cycling. In contrast, less than 10% of both day-8 and day-12 CFU-S are killed by S-phase-specific agents. From this we conclude that a high uptake of Rh 123 depends on other factors in addition to the cell cycle status. It is suggested that differentiation processes in which the day-8 CFU-S appears to be involved also cause the presence of many or very active mitochondria. The difference between the weakly and brightly fluorescing fractions in the number of day-12 CFU-S required for 30-day survival after lethal irradiation, suggests that there is heterogeneity among the day-12 CFU-S population or that the fraction weakly labeled with Rh123 contains other, less mature, cells that are responsible for survival after lethal irradiation. Finally, the presence of prothymocytes in the brightly labeled fraction shows that these cells are different from the stem cells that protect lethally irradiated mice.