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.