Tissuelyser 2

Islets were lysed directly after isolation or after culture for 24 hrs in home-made RPMI (S1 Table) with glucose and MgCl₂, as indicated. Islets were lysed by Qiazol (Qiagen, Hilden, Germany) using the TissueLyser II, and chloroform (Sigma, St. Louis, USA) phase separation was used to extract total RNA. Purification was performed according to the manufacturer’s protocol, including an on-column DNase (Qiagen, Hilden, Germany) digestion step to eliminate genomic DNA. RNA was eluted in two steps using pre-heated RNA-free water and the concentration was checked using a Nano-drop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, USA). Equal amounts of total RNA (0.1–1.5 μg) were reversed transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Lithuania) according to the manufacturer’s protocol. The cDNA was subsequently used to determine the mRNA levels of Trpm6, Trpm7, Slc41a1, Slc41a2, Slc41a3, Cnnm1, Cnnm2, Cnnm3, Cnnm4, Gck and Abcc8 in islets and Trpm7, Cacna1c, Cacna1d, Ins1, Kcnj11, Abcc8, and Gck in INS-1 cells using commercially available Taqman probes by the StepOnePlus Real-Time PCR System (Applied Biosystems) and normalized to Actb expression (Table 1). Data were analyzed using the Livak (2^-ΔΔCT) method.

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.

DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen). Frozen cecal samples (~ 60 mg) were lysed in LoBind Eppendorf tubes using a TissueLyser II (10 min, 30 Hz) following addition of 1 mL glass beads (acid-washed, 106 µm [-140 U.S. sieve], Sigma-Aldrich) and lysis buffer (InhibitEX buffer and proteinase K, Qiagen). DNA concentration was measured with the Qubit Quant-iT dsDNA High Sensitivity assay kit (Thermo Fisher Scientific Inc.). DNA quality was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc.) and by visual inspection following electrophoresis (1.2% (w/v) agarose gel in Tris–acetate-EDTA buffer).