Fast red tr 1 5 naphthalenedisulfonate salt

The iMSCs at passages 10, 20, and 30 were seeded in 6-well culture plates (1×10⁵ cells/well) and cultured in GM or OM. On day 14, in situ ALP activity was detected in cells stained with a solution containing 1.8 mM Fast Red-TR 1,5-naphthalenedisulfonate salt (Sigma-Aldrich) and 0.9 mM Naphtol AS-MX phosphate (Sigma-Aldrich). The culture plates were kept in incubators for 30 min, solution was removed, and samples were examined in a Leica MZ6 modular stereomicroscope (Leica Microsystems, Bensheim, Germany) attached to a digital camera (DFC310 FX camera, Leica Microsystems).
As described above, we immortalized primary MSCs and confirmed the gene and protein expression of TERT at passage 3 after cell transduction, which was considered “passage zero.” The following experiments were performed after this passage.

In order to detect in situ ALP activity, 0.8 ml of a solution of 1.8 mM Fast Red-TR 1,5-naphthalenedisulfonate salt (Sigma–Aldrich) and 0.9 mM Naphtol AS-MX phosphate (Sigma–Aldrich) was added in the wells with cells cultured on Ti-Nano and Ti-Micro. The plates were kept at 37°C for 30 min, the solution was removed and the Ti discs were dried overnight before examining by fluorescence in a microscope Axio Imager M2 (Carl Zeiss, Göttingen, GO, DE) equipped with an Axiocam MRm digital camera (Carl Zeiss). The images were processed using Adobe Photoshop CS5 Extended software (version 12.1 - Adobe Systems, San Jose, CA, USA). The in situ ALP was quantified by counting pixels on six discs of both Ti-Nano and Ti-Micro within three areas randomly selected in each disc (n=18).