Crizotinib Effects on Osteosarcoma Cells

Human osteosarcoma cancer cells U2OS transfected with α-tubulin-GFP construct were seeded in two µ- Slide 8 Well (10⁵ cells/well) (ibidi, Gräfelfing, Germany) each. Cells were kept overnight in the incubator at 37 °C and 5% CO₂, then they were treated with different concentrations of crizotinib (0.5 × IC50, IC50, 2 × IC50) or DMSO (negative control) and placed for 1 h on ice. One of the µ-Slide 8 well was stained directly after incubation on ice and the other µ-Slide 8 well was incubated at 37 °C and 5% CO₂ and stained after 24 h. Cells were washed with PBS and then stained with Hoechst 33342 Nuclear Stain (BioVision, Wiesbaden, Germany) at room temperature in the dark for 30 min. Subsequently, cells were washed with PBS to remove excessive Hoechst stain and mounted with Fluoromount-G^®. A AF7000 widefield fluorescence microscope (Leica Microsystems, Wetzlar, Germany) was used to perform the imaging. GFP was detected at 470 nm excitation and 525 nm emission. Hoechst stain was detected at 470 nm excitation and 447 nm emission. Images were analyzed using Fiji ImageJ software [61 (link)].