Alkaline phosphatase assay kit

The cells were scraped into Alp lysis buffer (10 mM Tris-HCl, pH 8.0; 1 mM MgCl2; 0.5% Triton X-100 in PBS). The samples were sonicated for 5 min. The Alp activity was measured with Alkaline Phosphatase Assay Kit (cat# MAK447, Sigma).

Osteoblast differentiation on the titanium discs was investigated using an ALP assay, which measures the activity of ALP as an osteogenic marker expressed by early osteoblast during bone formation. The titanium discs were placed overnight in the presence or absence of 10 μg·mL⁻¹ FN9-10_ELP at 4°C. Then, hMSCs were plated at a density of 5 × 10³ cells/disc and incubated for 5 and 10 days at 37°C. Next, the discs were rinsed with DPBS and the cells on the discs were lysed with 100 μL 0.1% Triton x-100 at RT for 30 min. After removing the insoluble materials by centrifugation at 4°C for 10 min, the ALP activity of the soluble cell lysate was evaluated using an alkaline phosphatase assay kit (Sigma). The absorbance of the colored reaction mixture was read at 405 nm using a microplate reader, and the ALP activity was normalized to the control (i.e., the non-coated titanium discs).

ALP activity was assayed with the use of Alkaline Phosphatase Assay Kit, Sigma86C. At culture day 7 cells were assayed for ALP activity. Briefly, following MTS test, cells were rinsed 3x with PBS. Then, 200 μl of the cell digestion buffer containing Cell Assay Buffer stock solution, composed of 1.5 M Tris, 1 mM ZnCl₂, MgCl₂•6H₂O, diluted 1:10 in dH₂O and 1% of Triton X-100. was added to each well in 24-well plate and the cells were kept at 4°C overnight. The following day cells were incubated for 30 min at 37°C. The cell lysates were transferred into clean centrifuge tubes, vortexed and centrifuged. To assay for ALP activity, 900 μl of ALP substrate solution (i.e., 37.1 mg of pNPP in 20 ml of Cell Assay Buffer, prepared as described above, was combined with 100 μl of the cell lysate. Following gentle mixing and incubation for 10 min at room temperature, changes in A_{405 nm} were measured over 6 min at 1-min intervals. The obtained values were next normalized against the number of cells obtained from the cell viability assays (MTS test), as described above.

ALP levels were detected using an alkaline phosphatase assay kit (Sigma‒Aldrich). The assay was conducted according to the manufacturer’s instructions. Briefly, 20 µl of cell culture supernatant was transferred into 96well plates. ALP working reagent, 180 µl, was added to the sample wells and the plate was tapped gently to mix. Absorbance was read at 405 nm.

Fetal bovine serum (FBS), minimum essential medium alpha medium (α-MEM), penicillin-streptomycin, and trypsin-EDTA were purchased from GIBCO (Grand Island, NY, USA). Dulbecco's Modified Eagle medium (DMEM) was obtained from Thermo Fisher Scientific (Pittsburgh, PA, USA). Cordycepin, alizarin red S, an Alkaline Phosphatase Assay Kit, and TRAP Staining Kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). RANKL was purchased from R&D systems (Minneapolis, MN, USA). 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from DUCHEFA Biochemie (Haarlem, The Netherlands). Mouse antibodies BMP-2, Runx-2, Osterix, Osteopontin, and sRANKL were purchased from Abcam (Cambridge, UK). Antibodies of mouse TRAF6, cFOS, phospho-cFOS, NFATc1, cathepsin K, p38 MAPK, phospho-p38 MAPK, ERK1/2, phospho-ERK1/2, JNK, and phosphor-JNK were provided by Cell Signaling Technology (Beverly, MA, USA). The anti-mouse HRP-conjugated secondary antibody and anti-rabbit HRP-conjugated secondary antibody were purchased from Enzo Biochem (Farmingdale, NY, USA). TOPreal™ qPCR 2X PreMIX and M-MLV cDNA Synthesis Kit were obtained from Enzynomics (Dajeon, KOR). PCR primers (OSCAR, Ctsk1, NFATc1, GADPH) were purchased from Qiagen (Hilden, DEU).

The in vitro osteocyte‐osteoblast co‐culture model was also divided into three groups: control, Ti and Ti + SOST‐shRNA group. Cells of the control group underwent conventional culture, while osteocytes of the Ti and Ti + SOST‐shRNA group were treated with 1 mg/mL of Ti particles. ALP activity in the co‐culture supernatant was measured on day 7 after Ti particles were added. For such, medium was collected and centrifuged twice at 4000 g for 10 minutes in order to remove cell debris and Ti particles. ALP activity was evaluated using an Alkaline Phosphatase Assay Kit (Sigma‐Aldrich): assay mixtures contained 2‐amino‐2‐methyl‐1‐propanol, MgCl2, p‐nitrophenyl phosphate disodium and cell homogenates. After incubation, the reaction was stopped with NaOH and absorbance was read at 405 nm.
The cell co‐culture was maintained as described above. Similarly, ALP staining was performed on day 7 after Ti particles were added. Cells were washed three times with PBS prior to staining with an Alkaline Phosphatase Stain Kit: cells were fixed in methanol and overlaid with 5‐bromo‐4‐chloro‐3‐indolyl phosphate plus nitroblue tetrazolium chloride in Tris‐HCl, NaOH and MgCl2, followed by incubation at room temperature for two hours in the dark.

Retroviral particles encoding the Yamanaka’s factors were used to infect primary keratinocytes obtained from neonate mice in the presence of polybrene (8 µg/ml) and, after 2 days, transferred to an iPSC medium composed of DMEM-GlutaMAX (Thermo Fisher Scientific, Catalog No. 31966-021), 15% knockout serum replacement (Thermo Fisher Scientific, Catalog No. 10828-028), 1x nonessential amino acids (Thermo Fisher Scientific, Catalog No. 11140-035), 100 µM 2-mercapthoethanol (Thermo Fisher Scientific, Catalog No. 31350-010), and 1 × 10³ u/ml LIF (Millipore, Catalog No. ESG1107). Three days later, cells were collected using Acutase and seeded on feeder cell layers generated as described elsewhere^{76 (link)}. iPS cell colonies were allowed to grow for two weeks, with daily medium changes, and finally stained with an alkaline phosphatase assay kit (Sigma-Aldrich, Catalog No. AB0300) according to the manufacturer’s instructions. The generation of bona-fide iPS cells was further confirmed using qRT-PCR analysis to detect Nanog expression.