2.8. Alizarin Red S staining assay
To characterize the mineralization of BMSCs after EV treatment an Alizarin Red assay was conducted. Cells were cultured for 7, 10, 14, and 21 days with EV treatment before being washed and fixed as in section 2.3. After fixation, the cells were washed with distilled water and incubated with Alizarin Red solution (pH 4.0–4.3) for 5 min. The extra dye was removed by rinsing with distilled water three times. Samples were observed using light microscopy (Leica DM4000 B). To quantify calcium deposition, 200 μL of 10% acetic acid was added to each Alizarin Red stained-well and shaken for 30 min. The cells were then collected and transferred into a microcentrifuge tube where they were vortexed for 30 sec. They were then heated to 85°C for 10 min before being cooled on ice for 5 min. The slurry was centrifuged for 15 min and the supernatant moved to another tube where it was neutralized with 10% ammonium hydroxide. This was then added to a 96-well plate and the absorbance was read on a plate reader (SpectraMax M5 Microplate Reader). The concentration was compared to a standard curve for quantification. The Alizarin Red staining assay was performed at 7, 10, 14, and 21 days.