Liquicolor kit

For measurements of Ca⁺⁺, blood was obtained from the tail vein, collected in a capillary tube, and immediately processed using the RapidLab 348 Ca⁺⁺/pH analyzer (Siemens, Germany).
For other serum biochemical parameter measurements, blood was obtained by cheek bleeding, collected into the CAPIJECT T-MG micro collection tube (Terumo Europe N.V., Belgium), and centrifuged to yield serum that was stored at −80°C until further analysis. Samples were analyzed for phosphate using Phosphor C kit (Wako Pure Chemical Industries, Osaka, Japan), for PTH using mouse PTH 1-84 Elisa kit (Immutopics, San Clemente, CA), and 1,25-dihydroxy vitamin D₃ (1,25(OH)₂D₃) using 1,25-Dihydroxy Vitamin D EIA kit (IDS, Fountain Hills, AZ).
Spot urine was collected every 2 to 4 hours over 24 hours and was stored at −20°C until further analysis. Urinary calcium and creatinine concentrations were determined using the LiquiColor kit (Stanbio Laboratory, Boerne, TX). Urinary calcium concentration (uCa) was expressed as the ratio to the urinary creatinine concentration (uCa/uCr). Urinary cAMP production was measured by RIA as described [8 (link)].

Non-esterified fatty acid levels in serum were determined using Wako Chemicals lipid assay systems (Wako Diagnostics, Richmond, VA). Colorimetric analyses of lipids were measured at 570 nm on an Omega FLOUstar 96-well plate reader from BMG labtech (Ortenberg, Germany). Serum β-hydroxybutyrate measurements were determined from mouse serum samples using a β-hydroxybutyrate LiquiColor kit (Stanbio Laboratory, Boerne, TX, USA) according to the manufacturer instructions.

5×10⁴ C2C12 cells were seeded into 24-well plate and incubated overnight at 37 °C and 5% CO₂. GF/MSN, GF/MSN/CTS-DFO, GF/MSN-BMP2 and GF/MSN-BMP2/CTS-DFO scaffolds were then added to each well. After 7 days of culture, ALP activity was carried out using an EnzoLyte pNPP Alkaline Phosphatase Assay Kit (AnaSpec, San Jose, CA), as was previously described [47 (link)] with some minor modifications. ALP activity was measured at 405 nm and normalized against total protein content. The total protein content was measured with a BCA kit ( Thermo Scientific^™, Waltham, MA) according to the manufacture’s instruction.
The samples were also examined for calcium deposition by using a total calcium LiquiColor^® kit (Stanbio laboratory, TX). After 3 weeks of culture, cells were rinsed with PBS. The calcium was extracted by using 1 mL 6 M hydrochloric acid for 6 h. Thereafter, 10 μL extraction solution or 10 μL standard solution was added into 1 mL working solution prepared according to the manufacturer’s instruction. The absorbance was measured at 550 nm, and the calcium content was calculated from the following equation:
$$\mathit{Calcium}(mg/dL)=\frac{Au}{As}\times 10$$ where Au and As are the absorbance values of sample and standard, respectively.