2 amino 2 methyl 1 propanol buffer

L-aspartic acid (Sigma-Aldrich, UK), 1,4-diaminobutane (DAB) (Sigma-Aldrich, ≥99%), cysteamine (CYSE) (Sigma-Aldrich, UK), cystamine (CYS) (Sigma-Aldrich, UK), dimethylformamide (DMF) (VWR International, USA), dimethylsulfoxide (DMSO) (Sigma-Aldrich), o-phosphoric acid (VWR), imidazole (ACS reagent, ≥99%, Sigma-Aldrich), citric-acid*H₂O (ACS reagent, ≥99.9%, VWR), sodium chloride (99–100.5%, Sigma-Aldrich), phosphate buffer saline (PBS) (Tablet, Sigma), D,L-dithiotreitol (DTT) (Sigma), 5,5 dithio bis-(2-nitrobenzoic acid) (Sigma, ≥98%, USA), L-cystein (Sigma, ≥97%, USA), Humidified incubator (Nuaire, USA), 100 mm tissue culture dishes (Orange Scientific, Belgium), 48 well plates (Sigma-Aldrich, USA), low cell binding 96 well plates (Nunc, Denmark), Eagle’s Medium Alfa minimal essential medium (αMEM) (Gibco, USA), fetal bovine serum (FBS, Gibco, USA), L-glutamine (Gibco, USA), penicillin and streptomycin (Gibco, USA), L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA), beta-glycerophosphate (Sigma-Aldrich, USA), dexamethasone (Sigma-Aldrich, USA), WST-1 [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (Roche, Switzerland), Vybrant DiD (Molecular Probes, USA), 2-Amino-2-Methyl-1-Propanol buffer (Sigma-Aldrich, USA), Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System (Sigma-Aldrich, USA).

To quantify the ALP activity in the samples, we used a colorimetric method based on the conversion of p-nitrophenyl phosphate to p-nitrophenol in the presence of ALP. We lysed samples cultured with the extracts and with osteogenic medium with 200 μl of 0.1% Triton X-100 in 1xTE buffer (Sigma-Aldrich, Sant Louis, USA), followed by three freeze–thaw cycles. Subsequently, 50 μl of sample were combined with 50 μl of a mixture in 1:1:1 ratios of 1.5 M 2-amino-2-methyl-1-propanol buffer (Sigma-Aldrich, Sant Louis, USA), 20 mM phosphatase substrate solution (Sigma-Aldrich, Sant Louis, USA) and 1 mM MgCl₂. The samples were incubated for 30 min at 37 °C, and we stopped the reaction using 1 M NaOH. The production of p-nitrophenol was quantified by measuring the absorbance at 405 nm and comparing it against a standard curve prepared with known concentrations of p-nitrophenol. Results were then normalized to the results obtained in the proliferation assay. Cell proliferation was quantified by measuring the amount of dsDNA in the samples with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Waltham, USA) following the recommendations of the manufacturer. We mixed 100 μl of sample and 100 μl of a 1:200 dilution of PicoGreen reagent in a black 96-well plate.