Calcium assay kit

HAp nanoparticles were prepared using the W/O emulsion method, as described in our previous study. The starting materials included 80 mL of dodecane (CH₃(CH₂)₁₀CH₃) in the oil phase, 1.0 g of pentaethylene glycol dodecyl ether (C₂₂H₄₆O₆) as a non-ionic surfactant, and 2.5 M of calcium hydroxide. After stirring these reagents for 1 h at 50 °C, 1.25 M of potassium dihydrogen phosphate was added to these solutions. After 24 h, the product was centrifuged and washed with distilled water and ethanol to remove oil and surfactant. The resulting particles were dispersed in distilled water. Physicochemical characteristics were examined using XRD (D8 Advance, Bruker), FTIR (FT/IR-4100, Jasco), and TEM (H-7100, Hitachi). The product was filtered using a 0.45 µm filter to remove aggregated nanoparticles. The concentration of the filtered solutions was measured using a calcium assay kit in accordance with the guidelines of the manufacturer (BioAssay Systems). TEM was performed to observe the size and shape of HAp nanoparticles at an acceleration voltage of 75 kV. The particle size distribution of the HAp nanoparticles was measured via NTA (NanoSight NS10, Malvern).

Antibiotics/antifungicides (PSA) were purchased from Cultilab (São Paulo, SP, Brazil). All-trans retinoic acid, ascorbate, β-glycerophosphate, BMP-2, eosin, paraformaldehyde, ρ-nitrophenol phosphate (ρNPP), silver nitrate, and trypsin were obtained from Sigma-Aldrich (St. Louis, MO, USA). α-modified Eagle’s minimal essential medium (αMEM modified) and Dulbecco’s modified Eagle’s medium (DMEM) were acquired from Nutricell (São Paulo, SP, Brazil). DNAse, fetal bovine serum (FBS), SuperScript™ III, and Trizol^® were purchased from Invitrogen (Carlsbad, CA, USA). Calcium Assay Kit was obtained from BioAssay Systems (Hayward, CA, USA). SYBR^® Green PCR Master Mix was purchased from Applied Biosystems (Carlsbad, CA, USA). Immobilon-P membranes and the antibodies anti-β-actin (04-1116), -Smad1 (05-1459), -phosphorylated Smad1/5/8 (AB3848), -Smad4 (04-1033), BMP-٤ (MAB١٠٤٩), -BMP-٧ (MAB٤٣٥٠), -rabbit, and -mouse were obtained from Millipore (Danvers, MA, USA). Enhanced chemiluminescence Pierce ECL was purchased from Thermo scientific (Rockford, IL, USA). All chemicals were analytical grade.

Quantification of the intracellular calcium level was evaluated based on our previous report [62 (link)]. Briefly, cells were plated onto PDA/GO composite-coated 60 mm dishes (10–15 EBs per dish). Fourteen and twenty-one days after osteogenic induction, the intracellular calcium concentration was measured using a calcium assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s information. The optical density was read at 612 nm. The calcium level was expressed as mg/mg of protein.

Briefly, mineralized samples were dissolved into 0.5 M acetic acid (0.4 mL) overnight and quantified by the calcium assay kit (BioAssay Systems) in line with specific protocols.