Leucosep gradients

The use of blood from healthy male non-smoking volunteers was approved by the local ethics committee of the University of Giessen (No. 81/13). Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Leucosep gradients (Greiner Bio-One, Frickenhausen, Germany). CD14⁺ monocytes were enriched by positive selection using Dynabeads^® CD14 (Invitrogen, Karlsruhe, Germany) according to the instructions of the suppliers. Cell preparations were evaluated by flow cytometry (FACSCalibur^TM, Becton Dickinson, San Jose, CA, USA) using FITC-labeled monoclonal antibody M5E2 to CD14 (BioLegend, San Diego, CA, USA). Monocyte purity was above 75%. Cells were cultured for 3 h and stimulated with BzATP as described previously (15 (link)) and AAT-P (1 mg/ml) was applied together with BzATP.

All studies on primary human cells were approved by the local ethics committee at the University of Giessen (approval No. 81/13). Blood was drawn from according to their own statements healthy, male, non-smoking adult volunteers into sterile syringes containing 17.5 IU heparin (Ratiopharm, Ulm, Germany) per mL blood and PBMCs were separated on Leucosep gradients (Greiner Bio-One, Frickenhausen, Germany). In some experiments, LPS (5 ng/mL) was added to blood samples before gradient centrifugation. PBMCs were cultured for 3 h in 24-well plates at a density of 5 × 10⁵ cells/0.5 mL in RPMI 1640, 10% FCS, 2 mM l-glutamine. Non-adherent cells were removed, and cell culture medium was replaced by medium devoid of FCS. PBMCs were stimulated with BzATP in the presence or absence of β-NAD as described for U937 cells.

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.