Xds 2

Tumour spheroid formation was visually confirmed daily using an Optika XDS-2 trinocular inverse microscope equipped with a Camera ISH500, and their mean diameters were analysed using “ImageJ version 1.53.e” software (http://imagej.nih.gov/ij/). ImageJ is a free software that can be used for manually counting the cell numbers and calculating the cellular size (area / diameter). The ImageJ program was calibrated (set scale) using an image obtained from the same microscope with a known scale before it was used to calculate the cell size (in diameter). Following the calibration, the pictures of the tumourspheres were opened in the program, and a line was drawn across the diameter to measure the tumoursphere’s size. The diameters of the spheroids were measured at least three times to obtain the mean and standard deviation.

Control and differentiated cells were grown in 24 well plates. On a specific day (5th, 11th, 15th, 20th, 24th) after osteogenic medium application, cells were fixed by 4% paraformaldehyde (Cell Signaling Technology, Danvers, MA, USA) for 30 min at RT. After DPBS (Gibco, Paisley, UK) washing, formed Ca²⁺ deposits were stained by 2% Alizarin Red S (Merck Sigma-Aldrich, Darmstadt, Germany) for 3 min at RT. Next, the dye was aspired and cells were carefully washed three times by ultrapure water to remove the remaining stain. Finally, cells were kept in ultrapure water. Color intensity was monitored and images were captured by the inverted microscope XDS-2 (Optika). For quantification of total stained mineralized tissue, Alizarin Red S was eluted by destain solution (10% acetic acid, 20% methanol) for 15 min at RT [111 (link)]. The eluates were centrifuged (10,000× g) for 10 min at RT to remove mineral precipitates and cell debris. Then, the supernatant was transferred into 96 well plates in duplicates. The absorbance was measured at 405 nm using Synergy H1 Microplate Reader (BioTek, Winooski, VT, USA). The amount of mineral deposits is presented as optical density normalized to the total amount of proteins (mg), which were determined by BCA assay (Thermo Fisher Scientific).