Premix wst 1 solution

3-Aminopropyltriethoxysilane (APTES) as silane coupling agent, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and paraformaldehyde were purchased from Sigma Aldrich Chemical Company, USA and used as received. L-Glutamic acid and 25% glutaraldehyde solution was purchased from Junsei, Japan. Premix WST-1 solution was purchased from Takara, Japan. Calcein-AM and ethidium homodimer III staining solution were purchased from Biotium, USA. Dulbecco’s phosphate buffered saline (DPBS) solution, dulbecco’s modified eagle medium (DMEM) were purchased from Gibco. The mouse pre-osteoblast cells (MC3T3-E1) were purchased from Korea cells bank (Seoul, South Korea). The MC3T3-E1 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco), 1.0% penicillin G-streptomycin at 37 °C under 5% CO₂ atmosphere. The culture medium was changed in every 3 days. The nHA were synthesized as per details as given in our previous communication [14 (link)]. Cover glass (borosilicate glass type D 263® M) with a thickness of 0.13 to 0.16 mm and a radius with 15 mm was procured from Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany.

HT29 and HCT116 cells were cultured in 96-well plates (2 × 10⁴ cells/well) and maintained for 24 h. Then, the cell culture medium was replaced with McCoy’s 5A medium without serum and incubated at 37 °C with 5% CO₂ in a humidified atmosphere for 12 h. Subsequently, the cells were treated with 50 mM glucose (Wako, Osaka, Japan), 100 nM recombinant human insulin (Novo Nordisk Pharma, Tokyo, Japan), 250 µM OA (Sigma-Aldrich, Germany), 250 µM PA (Sigma-Aldrich, Germany)^{45 (link)}, 100 µM AICAR (AdipoGen, CA, USA), and phosphate-buffered saline as control (Ctrl) for 6 h, or siRNA for 72 h. After treatment, 10 µL of Premix WST-1 solution (TaKaRa, Japan) was added to each well and incubated for 6 h at 37 °C. After incubation, the absorbance was measured at 450 nm using a microplate reader (Benchmark Plus Microplate Reader, Bio-Rad, USA), at a wavelength of 690 nm.