Abstract
Hoechst side population (SP) analysis has proven to be a valuable technique for identifying and sorting stem and early progenitor cells in a variety of tissues and species. In this method, the DNA binding dye Hoechst 33342 is loaded into the cell population of interest; stem cells preferentially exclude this dye, and these low-fluorescence cells can be detected by flow cytometry. However, Hoechst SP analysis usually requires a flow cytometer equipped with an ultraviolet laser source for optimal performance. Unfortunately, ultraviolet lasers are expensive and are not common fixtures on flow cytometers. Violet laser diodes emitting in the 395- to 410-nm range are less expensive and have become much more common on flow cytometers, but do not provide optimal excitation of Hoechst 33342. DyeCycle Violet is a cell-permeable DNA binding dye with a chemical structure similar to Hoechst 33342, but with a longer excitation maximum. DyeCycle Violet can be substituted for Hoechst 33342 when performing side population analysis on a cytometer with a violet laser source. The procedure for DyeCycle Violet labeling for side population is described, as well as the limitations particular to this dye.