Mitras lb 940

The cells were seeded in a similar protocol as the one used for SEM imaging. After four weeks of incubation, the samples were washed two times with double-distilled water. 1 mL of 40 mM ARS (pH 4.1) was added to each well form a 96-well plate. The samples were incubated for another 20 min, at room temperature and then washed with double-distilled water, during shaking for 5 min. The quantification of the mineralized deposited in the cells seeded on the structures was done by extracting the calcified mineral at low pH, followed by neutralization with ammonium hydroxide and absorbance measurement at 405 nm using a Mitras LB 940 (Berthold Technologies) spectrophotometer.

5000 cells/ sample were cultured in complete MEM for 4 weeks under standard conditions of temperature and humidity. After this time, the culture medium was replaced with 16.67% MTS (Cell Titer 96^® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) and 83.33% MEM (5% FBS). After 2–3 h of incubation, the supernatant was collected and 100 µL from each sample was distributed in a 96-well plate. The absorbance was measured at 490 nm, using the Mitras LB 940 (Berthold Technologies, Bad Wildbad, Germany) spectrophotometer. The viability was calculated as percent from controls (non-stimulated samples i.e., 0 mT).

Mitochondrial Complex I activity was determined by a colorimetric assay (ab109721, Abcam) in mitochondria isolated from frozen myocardial tissues immediately before the test (Mitochondria isolation kit for tissues, ab110168, Abcam). The assay was based on the oxidation of NADH to NAD⁺ and on the simultaneous reduction of a dye, leading to increased absorbance at OD = 450 nm.
ATP content was determined in freshly collected and prepared protein extracts from snap-frozen tissues. (ATP Determination Kit, Molecular Probes, Eugene, OR) as previously described [26 (link)]. The assay is based on luciferase requirement for ATP in producing light (emission maximum ~560 nm at pH 7.8).
Samples were run in triplicate and luminescence was measured on a Mitras LB 940 (Berthold, Bad Wildbad, Germany).

Wnt reporter activity assay was performed as described previously (Glaeser et al, 2016) with some modifications. Wild‐type, Porcn^KO1.2, and Porcn^KO1.4 HEK293T cells were transfected in white, flat‐bottom 384‐well plates with 20 ng TCF4‐Firefly Luciferase, 10 ng Actin‐Renilla Luciferase and 20 ng of Wnt3A, Dvl3, or pcDNA3.1 plasmids. Dual luciferase readout was performed 72 h after cell seeding using the Mitras LB940 plate reader (Berthold Technologies, Bad Wildbad, Germany). TCF4‐Firefly reporter signal was normalized to β‐actin‐Renilla luciferase signal.

In order to measure the Human Osteocalcin protein, the cells were seeded similarly as for SE; the samples were prepared using Quantikine^®ELISA Human Osteocalcin Immunoassay (Catalog Number DSTCN0 (R&D SYSTEMS, Minneapolis, MN, USA)), according to the producer’s specifications. The standard Osteocalcin solution in the kit was used in order to obtain a standard curve for Osteocalcin calibration. A total of 50 µL from the supernatant of each sample was added to a 96-well plate, together with 100 µL of Assay Diluent. The samples were incubated while shaking during 2 h; after this time, they were washed 3 times using the washing buffer; 200 µL from the conjugate were added in each well. After another 2 h of shaking at room temperature, the samples were washed 4 times using the washing buffer; 200 µL from the Substrate solution was added to each well and then allowed to incubate for 30 min, in the dark. At the end of this period, the reaction was finished using 50 µL of the Stop solution. The osteocalcin protein secretion was measured spectrophotometrically at 450 nm with a correction at 570 nm, using the Mitras LB 940 (Berthold Technologies).