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Cancer stem cells (CSC) are increasingly recognized as key target cells for cancer therapy because they are both tumorigenic and chemoresistant and, therefore, can give rise to recurrent disease. Side population (SP) sorting using an ultraviolet (UV) laser is an established method to isolate CSC based on ABC drug transporter-dependent (e.g., ABCG2) Hoechst 33342 efflux. We here show that Vybrant® DyeCycle™ Violet (DCV), a DNA-binding fluorophor allowing SP sorting using a non-UV laser (such as violet laser), is transported via P-glycoprotein (PgP). Because PgP might be particularly abundant in multidrug-resistant cancer cells rather than bona fide CSC, investigators using DCV should be aware that this strategy might also detect PgP-expressing non-CSC.