Labassay alp kit

Tissue culture polystyrene dishes (TCPS, 35 mm) were purchased from Iwaki, Japan. Type I collagen derived from tilapia (Oreochromis niloticus) scale and porcine skin were generated from Taki chemical, Japan, and Nitta gelatin, Japan, respectively. Dulbecco's modified eagle medium (DMEM) and Triton-X-100 were bought from Sigma-Aldrich, USA. Fetal bovine serum (FBS), TypLE express, and 1x phosphate buffered saline (PBS, pH of 7.4) were all from Gibco, USA. Glycerol-2-phosphate disodium salt n-hydrate (β-GP), L-Ascorbic acid phosphate magnesium salt n-hydrate, 10% formalin neutral buffer solution, Alizarin red S powder, and LabAssay ALP kit were purchased from Wako, Japan. Pierce BCA protein assay kit was from Thermo scientific, USA. TRIzol was purchased from Invitrogen, USA. Chloroform, 2-propanol, and ethanol were from Nacalai Tesque, Japan. FastStart Essential DNA Green Master for real time PCR reaction was purchased from Roche, Switzerland.

Cells were harvested and lysed with 0.1% (v/v) Triton-X-100 in distilled water and the lysates were sonicated on ice (Bioruptor, Diagenode, Seraing, Belgium) for 10 minutes and then centrifuged at 12,000 rpm, 4°C for 15 minutes (Hitachi Koki, Chiyoda, Tokyo, Japan). The supernatant was analyzed with a LabAssay ALP kit (Wako) according to the manufacturer's instruction. Total protein was quantified with a BCA protein assay kit (Pierce). ALP production was normalized to total protein amount. Absorbance was read using iMark microplate reader (BIO-RAD, Hercules, California, USA) at 405 nm and 570 nm for ALP assay and protein quantification assay respectively.

Alkaline phosphatase (ALP) bioactivity was examined by ALP staining and ALP activity testing after 7 days. For ALP staining, the acid-etched Ti plates loaded with cells and exosome samples were fixed in 4% paraformaldehyde and stained using a BCIP/NBT ALP color development kit (Biyuntian, China), then washed with PBS and photographed. ALP activity was determined using the LabAssay ALP kit (Wako Pure Chemical Industries, Japan) according to the manufacturer’s instructions. Total cellular proteins were determined using the BCA protein assay and the absorbance at 405 nm was measured with a Microplate reader (Thermo Fisher Scientific).

A solution (100 μL) containing GelMA (7.5% w/v), different amounts of OCP (0, 10, 20, 30% w/v), and the LAP photoinitiator (0.1% w/v) in the osteogenic differentiation medium (DMEM supplemented with 10% FBS, 1% PS, 50 μg/mL ascorbate 2-phosphate, 10 mM β-glycerophosphate, and 100 nM dexamethasone) were added into 96-well plates (Corning). Photopolymerization was carried out by a digital light processing (DLP) projector (Vivitek, Fremont, CA, USA) for 5 min. C3H10T1/2 cells were seeded on the GelMA hydrogel with or without OCP at a cell density of 4 × 10³ cells/well in 96-well plates with 100 μL osteogenic differentiation medium. Cells were cultured for 14 days at 37 °C, 5% CO₂ in humidified incubators. The culture medium was changed every two days. After 14 days of culture, cells were immersed in 0.1 mL of 0.2% Triton X-100 solution and sonicated on an ice bath. Then, cell lysates were centrifuged at 5000× g for 5 min to separate from the gel. DNA concentration of the supernatant was measured using a Quant-iT PicoGreen dsDNAkit (Thermo Fisher). Alkaline phosphatase (ALP) activity was measured using a LabAssay ALP kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The ALP activity was normalized using DNA amounts as determined with the Pico Green kit.

The HCEMs were seeded at a density of 1 × 10⁵ cells/well and cultured in MEMα containing 10% FBS. Twelve hours after seeding the cells, the culture medium was changed to an OS medium, and each cement specimen was placed on Transwell filter inserts with a pore size of 8.0 µm. The alkaline phosphatase (ALP) activity was measured after 7 days, using a p-nitrophenylphosphate assay kit (LabAssay ALP Kit; Wako Pure Chemical, Osaka, Japan). After 15 min of incubation at 37 °C, the absorbance of p-nitrophenylphosphate was determined using a microplate reader at 405 nm (Bio-Rad iMark TM; BIO-RAD Laboratories, Hercules, CA, USA), and the specific activity of ALP (U/µL) was calculated. Simultaneously, the cells were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical, Osaka, Japan; PFA) in PBS for 10 min at room temperature and stained using a nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt stock solution (Sigma-Aldrich, Saint Louis, MO, USA), based on the manufacturer’s instructions. The relative staining intensity was analyzed semi-quantitatively using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

ALP activity was measured by LabAssay™ ALP kit according to the supplier's instructions (Fujifilm Wako Pure Chemical Corporation). In brief, after two washes with PBS, cells were lysed with the help of sonication for 10 s in a buffer containing 0.1 m carbonate, 2 mm MgCl₂ (pH 9.8) and 0.3% Nonidet P‐40. Cell lysates were applied to ALP reaction using p‐nitrophenylphosphate, an ALP substrate, for 15 min at 37 °C, followed by measuring the absorbance at 405 nm (A_405 nm) with the microplate reader (Mithras LB 940; Berthold Technologies GmbH & Co.KG, Bad Wildbad, Germany). Protein concentration in the lysate was determined using Protein Assay Bradford Reagent (Fujifilm).