We performed the alkaline phosphatase activity assays using a kit (K412-500, BioVision, Inc., Milpitas, CA, USA) on Days 1, 5, and 7 following the manufacturer’s protocol. In short, the cell spheroids were suspended in an assay buffer, sonicated, and centrifuged. Then the supernatant was mixed with a p-nitrophenylphosphate substrate and incubation was performed. The optical density of the resultant p-nitrophenol was measured spectrophotometrically at 405 nm. We performed Alizarin Red S staining on Days 7 and 14.
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