Fbs rpmi 1640

The ATCC‐authenticated lung cancer cells (A549, NCI‐H460, NCI‐H1299, NCI‐H358, NCI‐H1650, and NCI‐H661) were purchased from the cell bank of Shanghai Institutes for Biological Sciences (Shanghai, China). A549 is a lung carcinoma cell line with the KRAS mutation. NCI‐H358 is a human non‐small cell lung cancer derived from the metastatic site (alveolus) and it expresses protein and RNA of lung surfactant‐associated protein A. NCI‐H1650 and NCI‐H460 are human lung carcinoma cells derived from pleural effusion; NCI‐H460 highly expresses p53 mRNA as compared to normal lung tissue. NCI‐H661 and NCI‐H1299 are human lung carcinoma cells derived from the lymph node. NCI‐H661 expresses p53 mRNA, whereas NCI‐H1299 lacks p53 mRNA and protein expression. SPC‐A1 is lung adenocarcinoma cells with high expression of surfactant‐associated protein A. HBE135‐E6E7 is used as a normal bronchial epithelium cell line. All cell lines were cultured in 10% FBS RPMI 1640 (Biowest, France) with 5% CO₂ at 37°C. The cells at an exponential rate growth were used in our study.

Platelet-rich plasma (PRP) and peripheral blood mononuclear cells (PBMCs) were isolated from 24 Evolut recipients and 40 Portico recipients (20 #P + 20 #wP). PRP was obtained from 5 ml blood collected in sterile K₃EDTA vacutainer tubes by centrifugation at 180×g for 10 min. The supernatant, i.e., PRP, was then further centrifuged at 4000 rpm for 4 min at room temperature, and the pellet containing platelets was then processed for analysis by immunofluorescence and flow cytometry. When needed, a lysis buffer for red blood cells (1X RBC Lysis Buffer, eBioscience, Invitrogen, Carlsbad, CA, USA) was added to platelets and incubated on a shaker for 10 min at room temperature. After removal of PRP, the corpuscular blood component was diluted 1:3 with isotonic saline solution and stratified on Ficoll-Hypaque density gradient (Histopaque-1077®, Sigma-Aldrich, St. Louis, MO, USA) to obtain PBMC. After a twice washing, PBMCs were resuspended in 5% FBS-RPMI 1640 (Biowest, Nuaillè, France), counted, and stained for immunofluorescence. For experiments with valve discard fluids, PRP from 3 normal donors was incubated (1:1, v/v) with valve discard fluid, or isotonic saline solution as control, for 8 h at 37 °C in a 5% CO₂ humidified atmosphere. Then, PRP pellet was processed for platelet apoptosis assay (see “Flow Cytometry Analysis”).