Stem cell side population analysis and sorting using DyeCycle violet

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.

Figure 9.30.1

Typical optical configuration for DCV SP analysis (BD Biosciences LSR II), using 450/50 nm and 675/20 nm bandpass filters for blue and red signal detection, and a 580 long-pass dichroic to separate the signals.

Figure 9.30.2

Typical fluorescence distribution of premixed Spherotech 8-peak Rainbow beads and Invitrogen Molecular Probes InSpeck Blue beads, excited with a violet laser diode and detected through a 450/50 nm bandpass filter. Note that all microspheres are not distinguishable in this analysis; although these bead sets are not specifically designed for violet laser verification, their distinctive “signature” can be easily monitored over time and deteriorations in laser performance easily detected. Analysis was carried on a BD Biosciences LSR II (San Jose, CA).

Figure 9.30.3

Hoechst and DCV SP analysis of mouse bone marrow, using ultraviolet laser excitation (355 nm, 20 mW). Analysis was carried on on a BD Biosciences LSR II. Note the difference in distribution and orientation of the SP population with DCV in comparison with Hoechst 33342.

Figure 9.30.4

DCV SP analysis of mouse bone marrow and human cord blood, using either ultraviolet (355 nm, 20 mW) or violet laser diode excitation (405 nm, 25 mW).