The Minimal Instrumentation Requirements for Hoechst Side Population Analysis: Stem Cell Analysis on Low‐Cost Flow Cytometry Platforms

The Hoechst side population (SP) technique is a critical method of identifying stem cells and early progenitors in rodent, nonhuman primate, and human hematopoietic and nonhematopoietic tissues. In this technique, the cell‐permeable DNA‐binding dye Hoechst 33342 is loaded into the cell population of interest; stem cells and early progenitors subsequently pump this dye out via an ATP‐binding cassette membrane pump‐dependent mechanism, resulting in a low‐fluorescence “tail” (the SP) when the cells are analyzed by flow cytometry. This population contains stem cells and early progenitors. One significant drawback of this method is the requirement of an UV laser to excite the Hoechst 33342. Unfortunately, flow cytometers equipped with UV sources are expensive to own and operate and are not readily available to many laboratories or institutions. In the interests of designing a less expensive flow cytometric system for stem cell analysis, we determined the minimum UV excitation and instrumentation requirements for measuring Hoechst SP. Less than 3 mW of UV laser output was required for adequate resolution of Hoechst SP on two cuvette‐based flow cytometers, one of which was a simple, inexpensive benchtop analyzer (the Quanta Analyzer; NPE Systems). Furthermore, Hoechst SP could also be adequately resolved on this epifluorescence‐based cytometer platform using two nonlaser UV sources, a mercury arc lamp with a UV bandpass filter and a UV‐emitting light‐emitting diode. These results suggest that an economical flow cytometric system can be designed that is capable of resolving Hoechst SP, with a cost far lower than most UV laser‐equipped commercial systems. An inexpensive system of this type would make Hoechst SP analysis available to a much broader group of stem cell investigators.