Monocyte attachment medium

Peripheral blood mononuclear cells (PBMCs) (Vanderbilt Center for Immunobiology) isolated from fresh blood of three healthy anonymous donors were prepared from buffy coats using Ficoll-Paque (GE 45–001–749). Monocytes were enriched from PBMCs by Monocyte Attachment Medium (PromoCell 50306290). The monocytes were expanded in culture for 7 d in the presence of 100 ng/ml human GM-CSF. Cells were re-seeded onto 24-well plates at 1 × 10⁵ cells/well and polarized for 48 hr with the addition of 50 ng/ml human IFNγ (R&D systems) for M1 macrophages. These cells were subsequently treated with HT-DNA in the presence of Lipofectamine 2000 (Life Technologies) for an additional 16 hr in the absence of polarizing cytokines.

Human peripheral blood mononuclear cells (PBMC) were isolated from the peripheral venous blood of healthy volunteers. Briefly, whole blood (20 mL) was mixed with 7 mL of a 6% dextran solution and 15 mL of HBSS and allowed to stand for 30 minutes at 25 °C until stratification occurred. The upper leukocyte-rich plasma layer was transferred to a new tube containing endotoxin-free Ficoll-Paque PLUS gradient (GE Healthcare Japan, Tokyo, Japan) and was centrifuged (500 g, 30 min, 25 °C). The cell layer was harvested and subsequently, the cells were resuspended at 100 million PBMC per mL in Monocyte Attachment Medium (Promocell, Heidelberg, Germany). The cells were cultured for 10 days as per the manufacturer’s protocol and used for the phagocytosis assay, as described above. The protocol was approved by the Ethical Review Committee at the Teikyo University School of Medicine (no. 15-192-2). All participants gave a written informed consent prior to their inclusion in the study.

Peripheral blood mononuclear cells (PBMC) fractions were isolated from peripheral blood by density centrifugation with Lymphoprep (Axis-Shield PoC As, Oslo, Norway). Briefly, blood samples were diluted 1:1 with phosphate buffered saline (PBS, Gibco Invitrogen from Thermo Fisher Scientific, Kandel, Germany). Subsequently, 35 mL of diluted blood was added onto 15 mL of Lymphoprep, and centrifugated at 1200 rpm for 30 min at room temperature. Afterward, PBMCs portion was collected by aspiration with a Pasteur pipette and washed twice with PBS. The cell concentration was determined with a Neubauer hemocytometer chamber, discarding the dead cells by trypan blue exclusion test.
In order to isolate the monocytes from PBMCs portion, 30 × 10⁶ cells were seeded in 10 cm culture-treated dishes (Thermo Fisher Scientific, Kandel, Germany), adding 10 mL of Monocyte Attachment Medium (Promocell, Heidelberg, Germany) to promote monocyte adhesion. After 1.5 h of incubation at 37 °C and 5% CO₂, the supernatant was aspirated from the dishes and vigorously washed 3 times with PBS to eliminate nonadherent cells.

Experiments were approved by the Ethics Committee of the faculty of medicine, LMU Munich (18-821 and 21-0816), in accordance with the Declaration of Helsinki. Blood samples were collected after receiving the donors` written informed consents. MNC were isolated from peripheral blood or leukoreduction system chambers by density gradient centrifugation using Ficoll<sup>®</sup> Paque Plus (Cytiva, Marlborough, MA, USA). Monocytes were isolated by plastic adherence using monocyte attachment medium (PromoCell GmbH, Heidelberg, Germany) following the manufacturer`s protocols. To generate non-polarized M0 macrophages adherent monocytes were cultivated in X-Vivo™ 15 (Lonza) medium supplemented with 0.5% Pen/Strep and, unless otherwise indicated, M-CSF at a concentration of 50 ng/ml for 7 days. In individual experiments, M-CSF was replaced by GM-CSF (10 ng/ml). If not specified, polarized M1 macrophages were obtained by culturing cells in the presence of GM-CSF (10 ng/ml) for 6 days to drive macrophage differentiation towards an M1 phenotype, before stimulation with IFN-γ (10 ng/ml) and LPS (100 ng/ml) for additional 48 h. M2 macrophages (M2c subtype) were polarized with M-CSF (50 ng/ml) for 6 days, before IL-10 was added at a concentration of 10 ng/ml for additional 48 h. Macrophages were harvested by accutase (Thermo Fisher Scientific) treatment following the manufacturer`s recommendations.