Alizarine red s

To evaluate the osteogenic potential of the Feru-AFC NP-treated pMSCs and mMSCs in vitro, the calcium deposits were investigated by alizarin red S staining. Cells were seeded on a 24-well plate with 5,000 pMSCs/well and 10,000 mMSCs/well. The cells were then labeled by Feru-AFC NPs in the presence of lipofectin at a Fe concentration of 100 µg/mL for 24 hours. The labeling medium was then discarded and replaced by osteogenic differentiation medium, consisting of low-glucose DMEM supplemented with 10% FBS (Gibco), 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco), 10% L-glutamine (Gibco), 50 µg/ml L-ascorbic acid 2-phosphate sequimagnesium (Sigma), 100 µg/ml sodium pyruvate (Gibco), 0.1 µM dexamethasone (Sigma, St Louis, MO), and 100mM β-glycerophosphate. The osteogenic medium was changed every other day for 14 days. The cells were then washed with PBS buffer and fixed in 10% neutral buffered formalin for 15 min at room temperature. After washing with PBS, the fixed cells were stained with alizarine red S (Sigma) for 5 min and the reaction was stopped with DI water. The cells were further washed with DI water until the solution turned clear. Images were then taken using a microscope.

Alizaline red-S staining was carried out to determine the effects of whey protein hydrolysates on the formation of calcified nodules in MC3T3-E1 cells. The differentiated MC3T3-E1 cells were first treated with whey protein or whey protein hydrolysate. Cells were washed with saline, fixed with 70% ethyl alcohol, and stained with 40 mM (pH 4.2) Alizarine red-S (Sigma-Aldrich Co.) solution for 10 min. The area of calcified nodules was measured using an Image Analyzer (Media Cybernetics, Inc., Rockville, MD, USA) and expressed as a percentage compared with the normal group (Kawazoe et al., 2004 (link)).