Replacing Hoechst33342 with Rhodamine123 in isolation of cancer stem-like cells from the MHCC97 cell line

The side population (SP) is commonly applied to analyzing cancer stem cells (CSCs) biology. However, the Hoechst33342 (Hoechst) dye, which is used for SP isolation, is demonstrated to be toxic to cells. The aim of this study was to identify a dye that might replace or offer an alternative to Hoechst use. In the trial group, by FACS binding Rhodamine123 (Rho), Rholow (low Rho fluorescence) and Rhohigh (high Rho fluorescence) were sorted from the MHCC97 cell line. In the control group, SP (low Hoechst fluorescence) and NSP (high Hoechst fluorescence) were obtained through Hoechst/FACS. The percentages of subpopulations were as following: Rholow 2.1%, Rhohigh 97.9%, SP 1.4%, NSP 98.6%. The proliferative abilities in vitro (growth rate, soft agar clone formation rate), expressions of stem cell markers (early hepatic marker AFP and CSCs marker CD133), and tumorigenicities in vivo were significantly higher in the Rholow and SP cells than those in the Rhohigh and NSP cells, respectively (P < 0.05). In addition, each result was the same between the Rholow and SP cells (P > 0.05). Taken together, Rho/FACS may not be as effective as Hoechst/FACS for CSCs sorting, but given its low toxicity and cost, it may be a useful method for CSCs identification.