Gm csf

An overview of the applied experimental design is illustrated in Figure 1. Monocytes were isolated from human BCs either by positive or negative selection. The purity of CD14⁺ cells was analyzed by flow cytometry directly after isolation, as previously described [22 (link)]. After isolation, the cells were adjusted to a final concentration of 1 × 10⁶ viable cells/ml and seeded in 24-well cell-repellent plates (Greiner Bio-One, Leipzig, Germany). Both activated and naïve monocytes were investigated. The activation of monocytes was experimentally induced by GM-CSF (10 ng/ml, PAN-Biotech, Aidenbach, Germany), as described previously [6 (link),22 (link)], while naïve cells remained unstimulated. The monocytes were cultivated overnight for 16 h at 37 °C (95% humidity). Longer cultivation duration was associated with a loss of migratory capacity [22 (link)] and, therefore, was not investigated in this study. After cultivation, the monocytes were harvested and examined in various tests to analyze their functionality (adherence, metabolic activity, LPS response, migratory capacity). Furthermore, the supernatants were collected and stored at −20 °C until cytokine measurement. After isolation and culturing of monocytes, the cells’ number, size, and viability were evaluated using the CASY cell counter according to the manufacturer’s instructions [22 (link)].

HEK293T cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Corning), 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 2 mM L-glutamine.
Primary murine BMDCs were obtained from tibia and femur bone marrows of C57BL/6 mice and cultured in Iscove’s modified Dulbecco’s medium (IMDM, PAN-Biotech) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% granulocyte-macrophage colony stimulating factor (GM-CSF) containing supernatant derived from X63 cells (72 (link)). Medium was exchanged every 48 hours and BMDCs were used after 8 days to ascertain that ≥80% of the cell population expressed the DC differentiation marker CD11c.
Primary human moDCs were obtained from PBMCs isolated from blood of healthy donors via Ficoll density gradient centrifugation. CD14⁺ monocytes were isolated by magnetic-activated cell sorting (Miltenyi Biotech) and differentiated into moDCs for 5 days in serum-free CellGenix GMP dendritic cell medium (CellGenix) supplemented with 1,000 U/mL GM-CSF (Miltenyi Biotech) and 1,000 U/mL interleukin-4 (IL-4, Miltenyi Biotech). All cells were grown at 37°C and 5% CO₂ in humidified atmosphere.

HEK293T cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Corning), 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 2 mM L-glutamine.
Primary murine BMDCs were obtained from tibia and femur bone marrows of C57BL/6 mice and cultured in Iscove’s modified Dulbecco’s medium (IMDM, PAN-Biotech) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% granulocyte-macrophage colony stimulating factor (GM-CSF) containing supernatant derived from X63 cells (72 (link)). Medium was exchanged every 48 hours and BMDCs were used after 8 days to ascertain that ≥80% of the cell population expressed the DC differentiation marker CD11c.
Primary human moDCs were obtained from PBMCs isolated from blood of healthy donors via Ficoll density gradient centrifugation. CD14⁺ monocytes were isolated by magnetic-activated cell sorting (Miltenyi Biotech) and differentiated into moDCs for 5 days in serum-free CellGenix GMP dendritic cell medium (CellGenix) supplemented with 1,000 U/mL GM-CSF (Miltenyi Biotech) and 1,000 U/mL interleukin-4 (IL-4, Miltenyi Biotech). All cells were grown at 37°C and 5% CO₂ in humidified atmosphere.