We provide important cautions for the fluorescent probe-based assessments of cellular metabolism in C6 glioma CSCs isolated by the SP method, i.e. requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation, by demonstrating the ability of glioma SP cells to expel fluorescent probes and the unexpected effect of Hoechst 33342 on the fluorescence corresponding to JC-1 aggregates. The gene discussed is ABCG2; the disease is glioma.