Bicinchoninic acid colorimetric assay

The cells were washed with phosphate buffered saline and they were collected with Mammalian Protein Extraction Reagent (Thermo Scientific, USA) containing1% protease inhibitor cocktail (Thermo Scientific, USA). Bicinchoninic acid colorimetric assay (Thermo) was performed to determine protein concentration. Equal concentration of protein (30–50 μg/well) were run in the SDS-PAGE gels (10–12%) and transferred onto PVDF membranes. The membranes were incubated for 1 h with blocking buffer containing 5% Bovine Serum Albumin (HiMedia Laboratories) in 1X Tris-Buffered Saline (TBS) solution containing 0.1% Tween-20 (1X TBS-T). Membranes were then incubated overnight with specific primary antibodies, such as TGF-β RII, Collagen 1A1 (COLL1A1), ALR, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), α-SMA (Abcam, Cambridge, UK), E-cadherin, and rac 1 (Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing 6 times x 5 minutes each with 1X TBS-T, the membranes were incubated with corresponding secondary antibodies for an hour and was eventually visualized using ECL Western blotting substrate. The membranes were developed after exposing to photosensitive films. As a loading control, β-actin was used in all the experiments.

At delivery, placental samples were weighted and, after discarding blood residuals, a full thickness section (from both maternal and foetal side) was obtained and processed as follows: 500 mg were homogenised (Caframo technologies, Ontario, Canada) with 10% BSA‐SolutionA to isolate fresh mitochondria and immediately perform ex vivo oxygen consumption assays.37 The remaining tissue was immediately cryopreserved at −80°C and further homogenised at 5% (w/v) in Mannitol for the rest of mitochondrial analysis.
Additionally at delivery, 10‐20 mL of maternal peripheral blood and neonatal cord blood were collected in ethylenediaminetetraacetic acid‐tubes to isolate plasma, PBMC and CBMC by a Ficoll gradient.38 One aliquot of each sample was maintained in fresh conditions to assess ex vivo oxygen consumption. The remaining aliquots were stored at −80°C for mitochondrial analysis.
The usefulness of mononuclear cells for the study of mitochondrial dysfunction has been previously validated. It offers an accessible and non‐invasive approach and the possibility to find new potential biomarkers for diagnosis and prognosis.39, 40Protein content was measured through the bicinchoninic acid colorimetric assay following manufacturer's instructions (Thermo Scientific, Waltham, MA) to normalise experimental measurements.