Inverted digital camera

Matrix calcification was observed using alizarin red staining. Culture medium was aspirated and cell monolayer was washed twice with PBS. Cell was fixed with 10% formalin neutral buffer solution for twenty minutes; afterwards the cell monolayer was washed again by PBS. Alizarin red solution (ARS) (1% w/v, pH 4.1) was added gently not to disrupt the cell monolayer. After five minutes, the staining solution was removed and the cell monolayer was firstly washed by distilled water and subsequently washed thoroughly with PBS to remove the nonspecific background stain. Photographs were taken using a digital imaging system (Funakoshi, Tokyo, Japan) incorporating an inverted digital camera (Canon, Tokyo, Japan). The quantification of staining was conducted using Cetylpyridinium Chloride (CPC) extraction method. Briefly, after staining with ARS, CPC (10%, w/v, in distilled water) was added to each dish (2 mL/dish) and incubated for one hour at 37°C. Following incubation, the transparent CPC solution, which turned into purple, was diluted by fivefold in original CPC solution and transferred to a 96-well plate (200 μL/well) for absorbance reading (BIO-RAD) at 570 nm.

In the same manner, cells were cultured in 12-well plates to day 10 at the concentration of 5×10³/mL (Low concentration) and 1×10⁴/mL (High concentration). Cells were fixed using 10% neutral buffer formalin (Wako) for 20 minutes, then the cell monolayer was stained with Alizarin red S solution (1%, w/v, in water, pH 4.0; Wako). Photographs were taken using a digital imaging system (Funakoshi, Tokyo, Japan) incorporating an inverted digital camera (Canon, Tokyo, Japan).

MDPC-23 cells were inoculated at the number of 1.25 × 10⁴/well and cultured with MM in 12-well plates until day four. At day four, IM described above in cell culture were added to culture media. Calcific deposition of cells was visualized using alizarin red staining by day eight. Briefly, cell monolayer was rinsed once by PBS and fixed in 10% formalin neutral buffer solution (060-01667, Wako) for 20 min at room temperature. Thereafter, cells were washed once using distilled water and stained by alizarin red s solution (1%, pH 4.1, 011-01192, Wako) for about five minutes at room temperature. Staining solution was discarded and cell monolayer was washed by distilled water for 2 h. The stained mineralized nodules were photographed by an inverted digital camera (Canon) and quantified using cetylpyridinium chloride (CPC). The detailed quantification method was described elsewhere [18 (link)].