4.3. Mineralization Detection via Alizarin Red Staining

An established Alizarin red S staining method was used to detect and quantify the mineralization of mineral nodules observed on the osteoblast cells [47]. About 35,000 cells/well HOBs were seeded overnight in a 24-well plate with treatment media. Then, the cells were induced with treatment media containing (i) media only as the negative control; (ii) 100 µM inorganic pyrophosphate (positive control); and (iii) 10, 25, 50, and 100 µg/Ml protein oxHDL in 400 µL of media (final total volume) for 14 days. The media was exchanged every 4 days.

On day 14 of incubation, the media was removed, and the cells were washed three times with PBS. The cells were fixed by incubation with 100 µL of 4% formaldehyde (diluted in PBS) at 4 °C for 45 min. Next, the fixative reagent was removed, and the cells were washed with distilled water three times. Then, the cells were incubated with 1 Ml of 2% alizarin red (PH 4.2) for 20 min at room temperature with gentle shaking. The dye was removed, and the cells were gently washed twice with distilled water. Pictures of the cells were taken using an inverted microscope (Olympus, Tokyo, Japan).

In total, 800 µL of 10% acetic acid was added to each well and incubated at room temperature for 30 min with gentle shaking. The cells in each well were collected using a cell scraper, transferred into a 1.5 mL micro-centrifuge tube, and vortexed for 30 min. Then, the tubes were heated at 85 °C using a digital block heater for 10 min. The tubes were put on ice for 5 min. The tubes were then centrifuged for 15 min at 20,000× g. About 500 µL of the supernatant was transferred into a new micro-centrifuge tube. Then, about 200 µL of 10% ammonium hydroxide was added into each tube to neutralize the acidic solution. The standard curve of alizarin red was prepared by diluting the 4 Mm alizarin red in serial dilution. The Ph of each dilution was between 4.1 and 4.3. In total, 150 µL of the samples and standard curve dilution was transferred into a 96-well plate. The optical density (OD) was measured using a microplate reader (PerkinElmer 2030 Multilabel Reader VictorTM X5, Waltham, MA, USA) at a wavelength of 450 nm. The concentration of alizarin red obtained for each sample was calculated based on the standard curve.