These results demonstrated that fluorescently labeled COM and COD can be detected by flow cytometry and used for quantitative analysis.
Vero cells were incubated with the crystals at 4 °C, at which endocytosis is hindered but adhesion is unaffected confirmed that binding Ca Ox to kidney epithelial cells reached the maximum within several minutes.
Vero cells isolated from kidney epithelial cells of an African green monkey are one of the most commonly used mammalian continuous cell lines in research on kidney stones.
Thus, in the present study, COM and COD crystals of different sizes (50 nm, 100 nm, and 1 μm) were prepared and compared in terms of endocytosis pathway and internalization mechanism toward Vero cells to reveal the cytotoxicity mechanism of kidney stone formation.
This phenomenon may be due to several factors: 1) small-sized crystals exhibit higher surface area than that of large-sized crystals and follow the order of COM-50 nm (26.3 m; hence, crystals with high surface area can create abundant active sites for cell adherence; 2) number of small-sized crystals is higher than that of large-sized crystals under the same crystal concentration; 3) large-sized COM crystals cannot completely form a contact with the cell interface, resulting in reduced shear stress and weak adhesive force between the cells and the large-sized crystals(A) Mean fluorescence intensity (FITC-A) and fluorescence ratio of cells.
The percentage of cells with adhered crystals was quantitatively determined through flow cytometry.
Cells with positive FITC signals were directly counted and considered as containing adherent crystals. showed that approximately 6% of MDCK cells adhered to COM crystals (crystal size of approximately 10 μm) at 100 μg/m L.
We used an integer (COM-50 nm, COM-100 nm, COM-1 μm, COD-50 nm, COD-100 nm and COD-1 μm) to represent the crystal size for simplicity and convenience.
The crystal phase was detected by XRD and FT-IR characterization presented in our previous study(A) Morphological observation of nano-/micron-sized COM and COD crystals.