Ethylene diamine tetraacetic acid (edta)

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from healthy (self-reported) female and male non-smoking adult volunteers. The study was approved by the ethics committee of the medical faculty Giessen, Germany (No. 90/18) and performed in accordance with the Helsinki Declaration. Blood was drawn into sterile syringes containing EDTA (1 mM per ml blood; bioWORLD, Dublin, OH, United States). LPS (E. coli O26:B6; 5 ng/ml) was added to blood samples shortly before PBMC isolation and PBMCs were separated using Leucosep gradients (Greiner) as described previously (Hecker et al., 2015 (link)). Thereafter, PBMCs were cultured for 3 h in 24-well plates (Greiner) at a density of 0.5 × 10⁶ cells/0.5 ml in Monocyte Attachment Medium (PromoCell, Heidelberg, Germany). Non-adherent cells were removed, and fresh Sigma RPMI 1640 medium was added. Stimulation with BzATP in the presence or absence of pCF3-diEPP was done as described for THP-1 cells. After cell treatment, cells were spun down (500 g, 8 min, 4°C), the supernatants were collected and stored at −20°C.

The study was approved by the ethics committee of the medical faculty Giessen, Germany (No. 90/18) and performed in accordance with the Helsinki Declaration. Each volunteer gave written informed consent. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from blood obtained from self-reported healthy, non-smoking adult volunteers. Blood was drawn into sterile syringes containing 1 mM EDTA (bioWORLD, Dublin, OH, USA) per ml blood and PBMCs were separated on Leucosep gradients (Greiner Bio-One, Frickenhausen, Germany). Before gradient centrifugation, LPS (5 ng/mL) was added to blood samples. Thereafter, PBMCs were cultured in 24-well plates at a density of 1 × 10⁶ cells/mL in Monocyte Attachment Medium (PromoCell, Heidelberg, Germany) for 3 h. Non-adherent cells were removed, and cell culture medium was replaced by fresh RPMI 1640 medium (Sigma-Aldrich R8758). Stimulation with BzATP in the presence or absence of Aβ_1-42, agonists or antagonists of nAChRs was done as described for U937 cells.