Nc 250 cell counter

Blood was collected for peripheral blood mononuclear cell (PBMC) isolation (in S-Monovettes® K3 EDTA (Sarstedt) or equal), as well as for serum isolation (S-Monovettes® Serum (Sarstedt) or equal). The blood was diluted with an equal volume of PBS (Corning) containing 2% fetal bovine serum (FBS; Gibco; heat-inactivated; from Brazil). PBMCs were obtained by density gradient centrifugation using SepMate™-50 tubes (Stemcell Technologies) filled with Lymphoprep™ density gradient medium (Stemcell Technologies) according to the manufacturer’s instructions. After the isolation, the PBMCs were stained with Acridine Orange and DAPI (solution 18, Chemometec), counted using the NC-250 cell counter (Chemometec), frozen in FBS with 10% DMSO, and stored in liquid nitrogen for at least one week before further experiments. Blood collected for serum isolation was centrifuged 10 min at 2,000xg. Serum was transferred into Eppendorf tubes and frozen at -20°C until use for further experiments.

Fresh blood, buffy coats, or mononuclear blood cell concentrates were obtained from healthy volunteers at the Department of Immunology or from the ZKT Tübingen gGmbH. Participants gave informed consent, and the studies were approved by the ethical review committee of the University of Tübingen, projects 156/2012B01 and 713/2018BO2. Blood products were diluted with PBS 1× (homemade from 10× stock solution, Lonza, Switzerland), and PBMCs were isolated by density gradient centrifugation with Biocoll separation solution (Biochrom, Germany). PBMCs were washed twice with PBS 1×, counted with an NC-250 cell counter (Chemometec, Denmark), and resuspended in heat-inactivated (h.i.) FBS (Capricorn Scientific, Germany) containing 10% dimethyl sulfoxide (DMSO; Merck). Cells were immediately transferred into a -80°C freezer in a freezing container (Mr. Frosty; TFS). After at least 24 h, frozen cells were transferred into a liquid nitrogen tank and were kept frozen until use. For the experiments, cells were thawed in Iscove´s Modified Dulbecco´s Medium (IMDM) (+L-Glutamin + 25 mM HEPES; Life Technologies) supplemented with 2.5% h.i. human serum (HS; PanBiotech, Germany), 1× P/S (Sigma-Aldrich), and 50 µm β-mercaptoethanol (β-ME; Merck), washed once, counted, and used for downstream assays.