Rpmi 1640 glutamax medium

Blood was collected in ethylenediamine tetraacetic acid (EDTA)-containing Vacutainer® tubes (Greiner Bio-one GmbH, Kremsmünster, Austria), and centrifuged at 1800 g for 10 min at 21°C. The plasma was aspirated and discarded, and the PBMCs were poured onto a density gradient medium (Lymphoprep TM ; Alere Technologies, Oslo, Norway), centrifuged at 1172 g for 30 min at 24°C, washed twice in sterile phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA), and finally resuspended in sterile medium consisting of RPMI-1640 GlutaMAX medium (Lonza, Basel, Switzerland) supplemented with 20% heat-inactivated fetal calf serum (hFCS) (Sigma-Aldrich, St Louis, MO, USA) and 0•1% gentamicin (BI, Kibbutz Beit Haemek, Israel) (referred to as incubation medium). PBMCs were subsequently counted using the NucleoCounter® NC-100 TM system (ChemoMetec, Allerød, Denmark), according to the manufacturer's instructions. The purified PBMCs were then divided into 500 µl aliquots in cryotubes (Dacos, Esbjerg, Denmark), followed by addition of 500 µl sterile medium, yielding a final concentration of 25% hFCS and 10% dimethyl sulfoxide (DMSO) (Merck kGaA, Darmstadt, Germany). The cryotubes were inverted, placed in CoolCell® freezing containers (BioCision, San Rafael, CA, USA) and stored at -80°C for at least 24 h before they were cryopreserved.