Anti cd14 pe

Monocyte subset analysis was performed using FACS Calibur flow cytometer (Becton Dickinson, United States) with monoclonal antibodies: anti-CD14-FITC, anti-CD14-PE, anti-CD11b-FITC, anti-CD18-PE, anti-CD54-FITC, anti-CD36-FITC, anti-CD16-PE, anti-CD206-PE (IO test, Beckman Coulter, France). 50 μl EDTA whole blood was stained with 5 μl of the relevant antibodies for 20 min at room temperature. In order to reduce Fc-receptor mediated binding of test antibodies, 2.5 μl Human Seroblock reagent (BIO-RAD, United States) were added for 10 min at room temperature. Red blood cells were lysed with 250 μl OptiLyse C Lysing Solution (Beckman Coulter, France) before analysis.

Three milliliters of PBMCs with a concentration of 5 × 10⁶ cells/ml was cultured in a well of a 6-well plate. After 1 h of incubation at 37°C and 5% CO₂, the nonadherent cells were washed 3 times with warm PBS. The remaining adherent monocytes were prestimulated for 24 h with concentrations of different ligands that would induce either training or tolerance, such as β-glucan (1 μg/ml), MDP (1 μg/ml), flagellin (1 μg/ml), R848 (1 μg/ml), Pam3CSK4 (1 μg/ml), and RPMI (negative control). The stimuli were washed away and the cells were further incubated for 5 days in the presence of RPMI supplemented with 10% pooled human serum. On day 7, the cells were incubated for 1 h with Versene solution, collected, harvested by centrifugation, and suspended in PBS supplemented with 1% protein blocking agent (PBA). The cells were washed two times and stained extracellularly with the following antibodies: anti-CD45-PeCy7 (Beckman Coulter), anti-CD68-APC (BioLegend), anti-CD14-PE (Beckman Coulter), anti-CD11b-FITC (Beckman Coulter), and anti-DC-SIGN-APC (BioLegend). The samples were measured on a fluorescence-activated cell sorter (FACS) FC500, and the data were analyzed using the CXP software (Beckman Coulter).

Detection of the cell surface expression of monocyte-derived myeloid cells was performed by flow cytometry using anti-CD1a-PE, anti-CD209-PE, anti-CD14-PE, anti-CD83-PE, anti-CD103-PE, anti-CX3CR1-PE, and anti-CCR7-PE (Beckman Coulter, Hialeah, FL, USA). The growth factor receptors were characterized by anti-GM-CSFRα-PE and anti-M-CSF R/CD115-PE (R&D Systems, USA) and isotype-matched control antibodies (BD PharMingen, San Diego, CA, USA). Fluorescence intensities were measured by FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed by the FlowJo software (Tree Star, Ashland, OR, USA). The human chemokines Mk, CXCL7, CCL20, and CXCL16 were ordered from PeproTech, UK; CXCL8 and CXCL1 are from Miltenyi Biotec.