Multiplex immunoassay kit

Maternal venous blood samples (7.5 mL serum tube, S-Monovette, Sarstedt, Nümbrecht, Germany) were obtained in a fasting state early in the morning. The samples were centrifuged at 4000 rpm for 10 min at 4 °C in a Hettich MR20 centrifuge (Hettich Lab Technology, Tuttlingen, Germany), and the serum was pipetted into new tubes for storage at −80 °C until evaluation.
Leptin levels were measured using either a direct sandwich enzyme-linked immunosorbent assay (ELISA) kit from MERCK/Millipore, Darmstadt, Germany, a TECAN reader from Nano Quant infinite M200 Pro, Switzerland, or a multiplex immunoassay kit from Bio-Rad Laboratories (Hercules, CA, USA) in accordance with the manufacturer’s instructions.
Resistin levels were measured using a commercially available multiplex immunoassay kit (eBioscience, San Diego, CA, USA or Bio-Rad) in accordance with the manufacturer’s instructions.

Human pulmonary artery endothelial cells (HPAEC) and human monocyte cell line (THP1) were obtained from American Type Culture Collection (ATCC). 2 × 10⁵ cells were seeded in 24-well plates and cultured in RPMI-1640 (10% fetal bovine serum, 100 U ml⁻¹ penicillin/streptomycin, 2 mM glutamine) in a 5% CO₂ humidified atmosphere at 37 °C. After the cells reached 80–90% confluence, they were incubated with 50% of the sera samples (approximately 400 μl) for 24 h, which were pooled from the patients before or within 24 h after transplantation, respectively. To verify the impact of extracellular histones in the sera, 20 μg ml⁻¹ of anti-histone H4 antibody or 200 U ml⁻¹ of heparin (Sigma-Aldrich, USA) was added to the cultured cells, respectively.
For the cytotoxicity assay, the cultured HPAEC cells were detached and washed with PBS and incubated with PI dye solution (10 μg ml⁻¹, Sigma-Aldrich, USA) in the dark for 10 min at room temperature. The cells were then subjected to flow cytometry analysis. In addition, the supernatants were collected and analyzed for LDH levels. To test the influence of extracellular histones in sera samples on THP1 cells, the supernatants (approximately 100 μl) were collected and analyzed for the production of multiple cytokines (IL-1β, IL-6, IL-10, IL-17A, IL-18, TNF-α) using a Multiplex Immunoassay kit from Affymetrix eBioscience.