Stepwise addition of DMSO.

<p><b>(A)</b> Comparison of the mortality rate of cells between stepwise and single-step addition. <b>(B)</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 <b>(A)</b>, the number of cells used was approximately 500 and the experiment was repeated 5 times. For <b>(B)</b>, the number of cells used was approximately 40. **p<0.01 was considered statistically significant.