Anti cd163 pe

Labeling was performed in PBS, EDTA 2 mM, fetal calf serum 5% at 4°C in 10⁵ cells. After the Fc receptor blocking step (FcR Blocking Reagent, Miltenyi Biotech), cells were stained with mixtures of monoclonal antibodies at the optimal concentration determined in our laboratory. A first fixation step was performed with 4% paraformaldehyde and a second with FoxP3/Transcription Factor Fixation Buffer (R&D Systems, USA). Intracellular FcRn expression was measured with A488 conjugated-FcRn antibody (Clone #937508 R&D Systems) by using the FoxP3/Transcription Factor Permabilization and Wash Buffer kit (R&D Systems) according to the manufacturer’s instructions. The following antibodies were used: anti-CD14 (PerCP-Vio 700, Miltenyi Biotech), anti-CD163 (PE, Miltenyi Biotech), anti-CD206 (PE-Vio 770, Miltenyi Biotech) and anti-CD16 (APC, Beckman Coulter, USA). Cell viability/apoptosis was detected using the APC Annexin V Apoptosis Detection Kit with 7-AAD (#640930, Biolegend, USA). Fluorescence was measured with a Cytoflex cytometer (Beckman Coulter). Data were analyzed with FlowLogic (Miltenyi Biotech). Flow cytometry analysis was performed in singlicate.

The following antibodies were used: anti-CD14-APC, anti-MHCII-DQ-PE, anti-MHCII-DR-APC (Immunostep, Salamanca, Spain) and anti-CD163-PE (Miltenyi Biotec, Auburn, CA, USA). The medium used for the cell culture was DMEM from Invitrogen. The LPS from Salmonella abortus was a kind gift from Dr. Galanos (Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany).