Anti cd163 apc

For in vitro macrophage induction, purified CD14+ monocytes were cultured in a complete RPMI medium (Life Technologies, Bleiswijk, The Netherlands) supplemented with 10% heat-inactivated calf serum (Merck Life Science, Madrid, Spain), 2 mM L-glutamine (Merck Life Science, Madrid, Spain), 1% antibiotic/antimycotic solution, and 100 ng/mL Macrophage Colony-Stimulating Factor (M-CSF) (Miltenyi Biotec, Bergisch Gladbach, Germany) for 8 days. Afterwards, M0 macrophages were polarized towards a M1 phenotype by incubating them with 120 ng/mL of IFNγ and 10 ng/mL of Lipopolysaccharides (LPS) (Merck Life Science, Madrid, Spain) for 48 h. For M2 phenotype, M0 macrophages were incubated with 2 µg/mL IL-4 (Peprotech, London, UK) for 48 h. MSCs were co-cultured with either M0, M1, or M2 macrophages at a ratio of 1:1. After 2 days macrophages were stained with the following antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany): anti-CD163-APC, anti-CD64-APC-Vio770, anti-CD209-PE-Vio770, anti-CD86-FITC, anti-MHC-I- PeVio770, anti-CD206 VioBlue, or with the corresponding isotype control antibodies. Data were acquired by flow cytometry using the MACSQuant^® analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Data were analyzed using MACSQuantify software (Miltenyi Biotec, Bergisch Gladbach, Germany) and FlowJo^TM v10.6.2 Software (BD Life Sciences, Ashland, OR, USA).

For intracellular cytokine detection, T cells were restimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL, Sigma-Aldrich) in the presence of Brefeldin A (0.2%, BD Biosciences) for 4 h. Cells were then stained with antibodies against T cell markers or their respective isotype control anti-CD25-PECy7 and anti-CD4-FITC (BD Biosciences) for 20 min at 4°C in the dark, fixed, and permeabilized with the Transcription factor buffer set (BD Biosciences) and then stained intracellularly with anti-Foxp3-PE, anti-IFNγ-PE, and anti-IL17A-AF647 antibodies (BD Biosciences) for 30 min at 4°C for flow cytometry detection of Tregs, Th1, and Th17 cells, respectively.
For flow cytometric detection of macrophages, cells were stained with the following antibodies or their respective isotype controls anti-CD40-APCy7 (BioLegend), anti-CD80-BV421 (BioLegend), anti-CD16-V500 (BD Biosciences), anti-CD206-AF488 (BioLegend), anti-CD163-APC (Miltenyi Biotec) for 20 min at 4°C in the dark. All samples were acquired on a MACSQUANT Q10 cytometer (Miltenyi Biotec) and analyzed using FlowJo v7.6.5 software (Tree Star).