Alp staining kit

ARS staining and ALP staining were conducted to examine the osteogenic activity. For ARS staining, hPDLSCs (2.5 × 10⁴ cells) were incubated for 14 days in the setting media containing osteogenic-inducing reagents. After 14 days, cells were fixed with 4% PFA, washed in PBS, and stained with 2% ARS solution (Acros, Gyeonggi-do, Korea). For ALP staining, hPDLSCs (2.5 × 10⁴ cells) were incubated for 3 and 7 days in the setting media including osteogenic-inducing reagents. After that, cells were fixed with 4% paraformaldehyde (PFA), washed in PBS, and stained using the ALP staining kit (Merck, Darmstadt, Germany). The ARS- and ALP-stained specimens were photographed using an optical microscope (Koptic, Gyeonggi-do, Korea), and digital camera (Nikon, Tokyo, Japan). The negative control groups (normal hPDLSCs) were cultured in normal media, while the positive control groups (osteoinducted hPDLSCs) were cultured in osteoinduction media at the same time as the experimental groups. These were stained and evaluated using the same protocol as above.

Alkaline phosphatase (ALP) staining was conducted to measure the osteogenic activity. hPDLSCs (2.5 × 10⁴ cells) were placed in a 24-well plate and incubated for 3, 6, and 9 days in the fresh extraction medium containing osteogenic-inducing reagents (100 µM l-ascorbic acid 2-phosphate, 10 mM b-glycerol phosphate, and 100 nM dexamethasone). After each time period, cells were fixed with 4% PFA for 10 min, washed twice in PBS, and stained using the ALP Staining Kit (Merck) according to the manufacturer’s recommendations.

To quantify hMSCs osteogenic differentiation, alkaline phosphatase enzyme activities were evaluated and measured by using two ALP staining assays, AP live staining (ThermoFisher) and ALP staining kit (for fixed cells, Sigma-Aldrich). For fixed cells, the staining solution was first prepared by mixing Fast Red Violet solution, Naphthol AS-BI phosphate solution and water at a ratio of 2:1:1. Next, hMSCs were fixed using 4% cold- Paraformaldehyde (PFA) for 2 min which enable the maintenance of the ALP activation. After fixation, the PFA was aspirated without wash. The staining solution was then added to the fixed cells for 15 min under room temperature and protected from light. The cells were then washed three times with 1x PBS, 15 min each time, before taking images. For AP live staining, hMSCs were stained using AP live stain at the concentration of 10X stock solution for 30 min according to manufacturers’ instructions. After staining, hMSCs were washed twice using basal medium. Images were captured after 30 min of staining. For F-actin staining, hMSCs were first fixed with 4% PFA solution for 10 min before being permeabilized and blocked with the PBST solution (PBS + 0.5% Triton + 1% BSA) for 1 h. After wash with 1x PBS three times, hMSCs were incubated with phalloidin (1:30) for 1 h at room temperature. The cells were then washed three times using 1x PBS, before imaging.