Eclipse ts2 136710

Ad-Cre SCs and Ad-Cre FAPs (2 × 10⁴) and eRMS cells were seeded in 6-well ultralow attachment microplates (Corning, USA) for 7 days. A DMEM/F12 serum-free medium (Invitrogen, USA #11330057) containing 5 μg/mL insulin, 20 ng/mL epidermal growth factor (EGF), 2% B27 and 20 ng/mL basic fibroblast growth factor (bFGF) was used to culture the spheres.
The total number of tumor spheres per plate was counted and images were acquired using an inverted microscope (Nikon, Amsterdam, Netherlands, model Eclipse Ts2 #136710).

Nitroblue Tetrazolium (NBT) assay was performed after 4 days of drugs treatment. Following treatment with different compounds, cells were collected, centrifuged at 300 × g for 7 minutes at room temperature and resuspended in complete IMDM growth medium or in alternative in RPMI growth medium at the density of 3.0 × 10⁵ cells/mL with NBT (Nitro blue Tetrazolium Chloride) (Sigma-Aldrich, #N6876) 1 mg/mL and MA (Phorbol 12-myristaPte 13-acetate) 5 µg/mL (Selleckem, # S7791). Next cells were incubated for 60 min at 37 °C. In differentiated cells NBT is phagosomed, the intracellular enzymes convert NBT into insoluble blue formazan crystals. At the end of the incubation cells were collected, centrifuged at 300 × g for 7 min and washed in PBS (Dulbecco’s Phosphate Buffered Saline, Biowest, #L0625-500). Next cells were resuspended in complete IMDM growth medium or in alternative in RPMI growth medium and seeded on 12 well plate (Falcon®, #353043). For each sample images were acquired by light microscopy using an inverted microscope (Nikon, model Eclipse Ts2 #136710). At least 200 cells for sample were counted using ImageJ software and the percentage of differentiated cells (cells containing blue-black formazan deposits) was calculated and processed using GraphPad Prism 7 software.