Sensolyte pnpp alp kit

Mesoporous silica nanospheres (MSNs), (3-aminopropyl) triethoxysilane (APTES), N-ethylcarbodiimide hydrochloride (EDCl), N-hydroxybenzotriazole (HOBT), type A gelatin (300 bloom) from porcine skin, methacrylic anhydride (MA), metformin hydrochloride (MF), and Alizarin Red were procured from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s phosphate-buffered saline (DPBS), alpha-modified Eagle’s Medium (α-MEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco-Invitrogen (San Diego, CA, USA). TRITC-conjugated phalloidin and DAPI were acquired from MilliporeSigma (Burlington, MA, USA). PureLink RNA Mini Kit, TRizol reagent, and the SuperScript II Reverse Transcriptase Kit were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP L0290, TCI America Inc., Portland, OR, USA), CellTiter 96 AQueous One Solution Reagent (Promega Corporation, Madison, WI, USA), SensoLyte pNPP ALP kit (AnaSpec Inc., Fremont, CA, USA), and Taqman real-time PCR Master Mix system (Applied Biosystems, Foster City, CA, USA) were procured from their respective manufacturers.

The colorimetric SensoLyte pNPP ALP kit (AnaSpec, Inc., Freemont, CA, USA) was used to evaluate the ALP activity according to the manufacturer’s recommendations. Cells were washed twice with 1× assay buffer. Then, cells were lysed in 1× assay buffer with 3 mL Triton-X-100. 50-μL supernatant was transferred to a 96-well plate after 10 min incubation at RT. A 50 μL pNPP was added to the supernatant and then allowed to react for 60 min at 25°C. The absorbance was measured at 405 nm. Total ALP activity was calculated based on an ALP standard of known concentration and was normalized to total protein.