Ficoll histopaque 1077 1

Leukocyte concentrates from freshly withdrawn peripheral blood of healthy adult human donors were provided by the Institute of Transfusion Medicine (University Hospital Jena, Germany). The experimental protocol was approved by the ethical committee of the University Hospital Jena. All methods were performed in accordance with the relevant guidelines and regulations. PBMC were isolated using dextran sedimentation and Ficoll-Histopaque 1077-1 (Sigma-Aldrich, Taufkirchen, Germany) centrifugation. PBMC were seeded in PBS containing 1 mM Ca²⁺ and 0.5 mM Mg²⁺ in cell culture flasks (Greiner Bio-one, Frickenhausen, Germany) for 1.5 h at 37 °C and 5% CO₂ for adherence of monocytes. For differentiation and polarization of monocytes towards M1- and M2-MDM, published criteria [27 (link)] were used. Thus, M1-MDM were generated by incubating monocytes with 20 ng/mL GM-CSF (Peprotech, Hamburg, Germany) for 6 days in RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine (Biochrom/Merck, Berlin, Germany), and penicillin-streptomycin (Biochrom/Merck), followed by 100 ng/mL LPS (Sigma-Aldrich) and 20 ng/mL IFN-γ (Peprotech) treatment for another 48 h. M2-MDM were obtained after differentiation of monocytes with 20 ng/mL M-CSF (Peprotech) for 6 days, and then with 20 ng/mL IL-4 (Peprotech) for additional 48 h of polarization.

Fetal bovine serum (FBS), RPMI 1640 culture medium, phosphate-buffered saline (PBS), and antibiotic solution (penicillin/streptomycin) were purchased from GIBCO (Gibco BRL Laboratories, Grand Island, NY, USA). Ficoll-Histopaque 1077-1, phorbol 12-myristate 13-acetate (PMA), and brefeldin-A (BFA) were purchased from SIGMA (St. Louis, MO, USA), Ionomycin from ENZO Life Sciences (USA), 7-AAD viability staining solution and reagents for cell staining, fixation and permeabilization as well all monoclonal antibodies used for flow cytometric analysis such as: anti-CD19-fluorescein isothiocyanate (FITC), anti-GM-CSF-phycoerythrin (PE), anti-CD3-FITC, anti-CD27-allophycocyanin (APC), anti-CD38-PE/Cy5 and appropriate isotype controls mouse IgG1-FITC, Rat IgG2α-PE, mouse IgG1-APC, and mouse IgG1-PE/Cy5 were obtained from Biolegend (CA, USA). Finally, the 96 rounded bottom well plates were purchased from Costar (Cambridge, MA, USA).

Human monocytes were isolated from de-identified leukopacks obtained from Children’s Hospital Blood Bank (Boston, MA) or the Institute of Transfusion Medicine, University Hospital Jena, Germany, with the use of Ficoll-Histopaque 1077-1 (Sigma-Aldrich, St. Louis, MO). Blood was obtained from healthy human volunteers giving informed consent under protocol #1999-P-001297 approved by the Partners Human Research Committee. The protocols for experiments with macrophages were approved by the ethical commission of the Friedrich-Schiller-University Jena. All methods were performed in accordance with the relevant guidelines and regulations. For differentiation and polarization towards M1 and M2, published criteria were used^{9 (link)}. Briefly, M1 was produced by incubating isolated monocytes with GM-CSF (20 ng/ml) for 6 days in RMPI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY), 2 mmol/l l-glutamine (Lonza, Basel, Switzerland), and penicillin–streptomycin (Lonza), followed by LPS (100 ng/ml) plus INF-γ (20 ng/ml) treatment for the indicated times (routinely 48 h). M2 was obtained by incubating monocytes with 20 ng/ml M-CSF for 6 days followed by polarization with 20 ng/ml IL-4 for the indicated times (routinely 48 h).

Human monocytes were isolated from de-identified leukopacks obtained from Children’s Hospital Blood Bank (Boston, MA) or the Institute of Transfusion Medicine, University Hospital Jena, Germany, with the use of Ficoll-Histopaque 1077-1 (Sigma-Aldrich, St. Louis, MO). Blood was obtained from healthy human volunteers giving informed consent under protocol #1999-P-001297 approved by the Partners Human Research Committee. The protocols for experiments with macrophages were approved by the ethical commission of the Friedrich-Schiller-University Jena. All methods were performed in accordance with the relevant guidelines and regulations. For differentiation and polarization towards M1 and M2, published criteria were used^{9 (link)}. Briefly, M1 was produced by incubating isolated monocytes with GM-CSF (20 ng/ml) for 6 days in RMPI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY), 2 mmol/l l-glutamine (Lonza, Basel, Switzerland), and penicillin–streptomycin (Lonza), followed by LPS (100 ng/ml) plus INF-γ (20 ng/ml) treatment for the indicated times (routinely 48 h). M2 was obtained by incubating monocytes with 20 ng/ml M-CSF for 6 days followed by polarization with 20 ng/ml IL-4 for the indicated times (routinely 48 h).

Leucocyte concentrates from freshly withdrawn peripheral blood of adult healthy donors were provided by the Institute of Transfusion Medicine of the University Hospital Jena, Germany. The experimental protocol was approved by the ethical committee of the University Hospital Jena. All methods were performed in accordance with the relevant guidelines and regulations. Neutrophils and monocytes were immediately isolated as described before [39 (link)]. In brief, cells were isolated by dextran sedimentation and Ficoll-Histopaque 1077-1 (Sigma-Aldrich) density centrifugation. To purify neutrophils, the remaining erythrocytes were removed by hypotonic lysis. Neutrophils were finally resuspended in PBS pH 7.4 plus 1 mg/mL glucose at the cell density of 5 × 10⁶ cells /mL. Monocytes were separated from peripheral blood mononuclear cells by adherence to cell culture flasks (Greiner Bio-one, Frickenhausen, Germany) for 1 h (37 °C, 5% CO₂), followed by cell scraping and resuspension in RPMI 1640 supplemented with 5% fetal calf serum, 2 mmol/L L-glutamine (Biochrom/Merck, Berlin, Germany), and 100 U/mL penicillin; 100 µg/mL streptomycin (Biochrom/Merck).