Ficoll paque density gradient separation

Blood was obtained from anonymized healthy SARS-CoV-2 negative non-vaccinated donors, upon informed consent and in agreement with the Declaration of Helsinki. The protocol was approved by the Regional Ethics Committee for Clinical Experimentation of the Tuscany Region (Ethics Committee Register n. 14,914 of May 16, 2019). Monocytes were isolated by CD14 positive selection with magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) from peripheral blood mononuclear cells (PBMC), obtained by Ficoll-Paque gradient density separation (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden). Monocyte preparations used in the experiments were > 95% viable and > 95% pure (assessed by trypan blue exclusion and cytosmears).
Monocytes were cultured in culture medium (RPMI 1640 + Glutamax-I; GIBCO by Life Technologies, Paisley, UK) supplemented with 50 µg/mL gentamicin sulfate (GIBCO) and 5% heat-inactivated human AB serum (Sigma-Aldrich). Cells (1x10⁵) were seeded in a final volume of 100 µL in 96-wells flat bottom plates (Corning^® Costar^®; Corning Inc. Life Sciences, Oneonta, NY, USA) at 37°C in moist air with 5% CO₂. Monocyte stimulation was performed after overnight resting.

Blood was obtained from healthy donors. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Regional Ethics Committee for Clinical Experimentation of the Tuscany Region (Ethics Committee Register n. 14,914 of May 16, 2019). None of the subjects was vaccinated with BCG or was positive in the tuberculin test. Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Paque gradient density separation (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden). CD14⁺ monocytes were isolated from PBMC using anti-CD14 antibody-bearing magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Cell viability was assessed by trypan blue dye exclusion and found to be > 95%. Monocyte purity was determined microscopically after cytocentrifugation and differential staining with a modified Wright-Giemsa dye (Diff-Quik; Medion Diagnostics, Duedingen, Switzerland). Only preparations with >95% purity were used.

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.