Tris hydroxymethyl aminomethane buffer ph 7.4

Osteochondral tissue constructs (N = 3, n = 3) were harvested and dissected, as previously described. The ALP activity and quantity of DNA was determined using a protocol modified from the literature [18 ]. The bone and cartilage ends of the tissue were homogenized separately using a TissueLyser II with ALP lysis buffer, consisting of 1 mM MgCl₂ (Sigma Aldrich, UK), 20 μM ZnCl₂ (Sigma Aldrich, UK) and 0.1% (w/v) octyl-β-glucopyranoside (Sigma Aldrich, UK) in 10 mM tris(hydroxymethyl)aminomethane buffer (pH 7.4) (Sigma Aldrich, UK) with the sample lysate immediately stored at −80 °C. To perform the assay, the samples were thawed on ice and then each sample was incubated with p-nitrophenol phosphate (Sigma Aldrich, UK) at 37 °C for 30 min. The reaction was terminated using 1 N NaOH and the absorbance measured at 405 nm using a SpectraMax M5 plate reader. A standard curve between 0 and 800 μM of p-nitrophenol phosphate was used to calculate the sample activity. An equivalent volume from the remainder of the sample was then used for DNA quantification using a PicoGreen™ assay (Thermo Fisher, UK), according to the manufacturer's protocol. The fluorescence was measured at 485/535 nm using a SpectraMax M5 plate reader. The ALP activity normalized to DNA quantity was compared between the bone and cartilage regions.