Cd14 magnetic microbead separation

Primary human monocytes were isolated from buffy coats of 20 healthy anonymous donors (provided by the blood bank of Salzburg, Austria, following overnight refrigeration), with cells from 4-8 buffy coats used for each primary stimulus. The study was conducted in accordance with the Declaration of Helsinki, and under Austrian national guidelines. According to Austrian regulations, no informed consent is required if blood cells derived from anonymous healthy donors, discarded after plasmapheresis (buffy coats) are used, therefore no additional approval by the national ethics committee was necessary. Peripheral blood mononuclear cells were obtained by Ficoll-Paque gradient density separation (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden). Monocytes were further isolated by CD14⁺ magnetic microbead separation (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instructions. The resulting cell suspension was monitored for purity by differential counting on Wright-Giemsa-stained cytosmears (Diff-Quik; Medion Diagnostics, Düdingen, Switzerland) examined by optical microscopy. Cell viability was assessed by trypan blue dye exclusion. Only cell isolations with at least 95% purity and 95% viability were used.