Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. Although SP abundance has been used as an indicator for disease prognostic and drug screening in many research projects, few studies have systematically examined the factors influencing SP analysis. In this study we aim to develop a more thorough understanding of the multiple factors involved in SP analysis including Hoechst 33342 staining and cell culture. RPMI-8226, a high SP percentage (SP%) human myeloma cell line was employed here. The results showed that SP% was subject to staining conditions including: viable cell proportion, dye concentration, staining cell density, incubation duration, staining volume, and mix interval. In addition, SP% was highest in day one after passage, while dropped steadily over time. This study shows that both staining conditions and culture duration can significantly affect SP%. In this case, any conclusions based on SP% should be interpreted cautiously. The relation between culture duration and SP% suggests that the incidence of SP cells may be related to cell proliferation and cell cycle phase. Maintaining these technical variables consistently is essential in SP research. 1. Introduction Side population (SP) cells were first described as a subset of adult mouse bone marrow with enriched hematopoietic stem cells (HSCs) [1, 2]. This subset was characterized by its ability to rapidly efflux the Hoechst 33342 DNA-binding dye and therefore shows a Hoechst profile on flow cytometry. Specifically they display a distinct staining pattern, based on the phenomenon of a differential emission of blue (450？nm) versus red (670？nm) emission fluorescence upon UV excitation, such that SP appears as a tiny population on the lower left-hand side of a red ( )-blue ( ) flow cytometry scattergram. This differential blue-red emission allows clear identification of a cell population that locates sideways from the diagonal and was thus named “side” population. Recent studies have shown the presence of SP cells in many types of cancer including ovarian cancer, glioblastoma cancer, lung cancer, nasopharyngeal cancer, gastrointestinal cancers hepatocellular carcinoma, mesenchymal tumors, and multiple myeloma [3–11]. SP cells in these types of cancer showed significantly higher potential to initiate tumor in NOD/SCID mice than their non-SP counterparts. They are also more likely to be resistant to certain anticancer drugs than non-SP cells. These results raised the significance of SP,
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