Stepwise addition of DMSO.
(A) Comparison of the mortality rate of cells between stepwise and single-step addition. (B) Bleb index in the stepwise addition method. HeLa cells were treated with 20% DMSO for 30 minutes, and the solution was removed quickly and changed to 40% DMSO for 30 minutes. It was then changed to 60% DMSO for 30 minutes and, finally, to 80% DMSO. The inverted fluorescence microscope was used to observe dead cells labeled by PI and Hoechst. For (A), the number of cells used was approximately 500 and the experiment was repeated 5 times. For (B), the number of cells used was approximately 40. **p<0.01 was considered statistically significant.