Digital dls camera

In order to evaluate matrix calcification, c643 stably overexpressing OPN-SV and TPC-1 cell lines were cultured in 24-well plates (1.5 × 10⁵ cells/well) for 31 days. Following 10, 17, 24, or 31 days in culture, cells were fixed with 4% paraformaldehyde (PFA) in PBS and evaluated for calcium deposits production by staining with 1% Alizarin Red solution in 2% ethanol. Cells were then washed three times with water, and representative images for each culture condition were captured with a NIKON microscope and NIKON Digital DLS Camera.

We cultured c643 overexpressing OPN-SV and TPC-1 cell lines in 24-well plates (1.5 × 10⁵ cells/well) for 31 days. Following 10, 17, 24, or 31 days in culture, cells were fixed with 4% paraformaldehyde (PFA) in PBS and evaluated for collagen fibers formation by Masson’s trichrome staining. Briefly, cells were immersed in Richard-Allan Scientific^® Bouin’s fluid (Thermo Fisher Scientific, Waltham, MA, USA) solution for 5 min at room temperature, washed in deionized water, followed by incubation in Celestin Blue (Thermo Scientific) for 6 min. Then, cells were incubated in Gil’s hematoxylin staining solution (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. Cells were washed with 1% acid alcohol and then underwent three sequential washes in deionized water. Cells were then immersed in Biebrich scarlet-acid fuchsin (Thermo Scientific) for 5 min, placed in phosphotungstic and phosphomolybdic acid solution (Thermo Scientific) for 5 min, moved to Aniline Blue solution (Thermo Scientific) for 5 min, and then placed in 1% acetic acid solution for 2 min. Finally, cells were rinsed in deionized water. Representative images for each culture condition were captured with a NIKON microscope and NIKON Digital DLS Camera.