Monocyte Isolation and Functional Analysis

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)].