Fxcycle pi rnase staining solution kit

Single cell suspensions of GSCs were prepared one day before treatment and treated with freshly prepared ClQ (Sigma-Aldrich, St. Louis, MO, USA, C6628-25G) at 30 µM/L for desired time points. For in vitro irradiation, cells were subjected to 2.5 Gy of X-rays using a Gulmay RS225 GS014 X-ray machine (Gulmay Medical Ltd., Camberley, UK) at a dose rate of 1 Gy/min. For flow cytometry, 2 × 10⁵ cells either untreated or untreated were harvested at indicated time points after treatment, washed in PBS and pelleted at 1500 rpm and +4 °C for 8 min. Washed cells were re-suspended in PBS, fixed with ice-cold 70% ethanol on the vortex and incubated for 15 min on ice. For staining, cells were rehydrated in PBS, stained using the FxCycle™ PI/RNAse Staining Solution Kit (Invitrogen) and processed for flow cytometric analysis of DNA content using the FACSCanto™Iia (BD, Franklin Lakes, NJ, USA) instrument. Statistical analyses were performed using the GraphPad Prism Version 7 (GraphPad Software Inc., San Diego, CA, USA). Self-renewal was assessed by using the extreme limited dilution assay (ELDA) [47 (link)].

Briefly, 2 × 10⁶ cells were harvested with trypsinization, washed, and fixed with 70% ethyl alcohol for 4 h under 4 °C, respectively. Then, cells were washed three times in 1 ml cold PBS. After dyed with Fxcycle PI/RNase Staining Solution kit (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature in the dark, the proportion of cells distributed in each phase was measured by BD FACSCalibur™ platform (BD, Becton, Dickinson and Company, Franklin Lakes, NJ, USA).