Pe conjugated anti cd163

The expression of surface markers and intracellular staining were assessed by flow cytometry. The following fluorescence-coupled antibodies were used in the study: fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (eBioscience, San Diego, CA, USA), FITC-conjugated anti-CD8 (BD Biosciences, San Jose, CA, USA), phycoerythrin (PE)-Cy7-conjugated anti-CD25 (BD Biosciences), and PE-conjugated anti-Foxp3 (eBioscience). APC-Cy7-conjugated anti-CD14 (BD Biosciences), BV510-conjugated anti-CD86 (BD Biosciences), PE-conjugated anti-CD163 (BD Biosciences), and FITC-conjugated anti-BAMBI (eBioscience) were also used. The relevant isotype controls were applied to confirm specific binding. Flow cytometry was performed on a BD LSRFortessa X-20 and analysed using FlowJo V10 software. CD14, CD86, and CD163 were assessed to validate the phenotypes of the macrophages; CD14 indicates the whole macrophage, CD86 indicates the M1 phenotype, and CD163 indicates the M2 phenotype. As previously described, CD4+ CD25+ FoxP3+ T cells represent Tregs.

Cell surface and intracellular cytokine staining of monocytes was performed as previously described (46 (link)). Briefly, cultured monocytes or purified monocytes were washed in PBS and stained by PE-conjugated anti-CD80 (BD Pharmingen, 557227), FITC-conjugated anti-CD86 (BD Pharmingen, 555657), PE-conjugated anti-CD163 (BD Pharmingen, 556018), FITC-conjugated CD206 (BD Pharmingen, 551135), PE-Cy7–conjugated HLA-DR (BD Pharmingen, 560651), or APC-conjugated anti-CD42b (BD Pharmingen, 551061) before analysis by FACSCanto. Fixation and permeabilization were performed using Cytofix/Cytoperm Plus kit with GolgiPlug (BD Biosciences) and stained by PE-conjugated anti–IL-1β (R&D Systems, IC201P) and PE-CF594–conjugated anti–IL-10 (BD Horizon, 562400) before analysis. All data acquired from flow cytometry were analyzed with FlowJo (Treestar software).