An in vitro study of Hoechst 33342 redistribution and its effects on cell viability

Although Hoechst 33342 (H342) is frequently used to label donor cells in cell transplantation research, it has been noted that it might secondarily label the host cells. Furthermore, its potential toxicity leading to cell death has been described. We studied the time course of H342 redistribution from the primary labeled rat bone marrow stromal cells (rBMSC) into the non-labeled rBMSC population over 7 days in culture; we evaluated the nuclear H342 fluorescence intensity as a possible criterion for distinguishing the primary from the secondary labeled cells, and determined the viability of rBMSC after an overnight incubation in 1 microg/mL of H342. H342 labeled >50% of the initially non-labeled cells within the first 6 hours and almost 90% within a week. Nuclear fluorescence intensity was a reliable criterion for distinguishing primary and secondary labeled cells within the first 24 hours, but less so at later time points. The percentage of either apoptotic or necrotic cells did not rise acutely after the overnight incubation in 1 microg/mL of H342. Although a 12-hour incubation of rBMSC in 1 microg/mL of H342 did not cause acute cell death, H342 rapidly and extensively redistributed into non-labeled cells, which makes H342 a relatively unsuitable marker for cell transplantation research.