Pe vio770 bdca2

Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats by Ficoll density centrifugation, from healthy donors (Etablissement Français du Sang, Paris Saint-Antoine-Crozatier, France) or untreated SSc patients (Hôpital Saint-Antoine, Paris, France) after informed consent. The study was approved by the local institutional review board and the Comité de Protection des Personnes Ile-de France VII (CPP Ouest-1, reference 2017-A03380-53). pDCs were negatively selected with EasySep™ Human Plasmacytoid DC Enrichment Kit (Stem cell, Grenoble, France). The purity of isolated pDCs, verified by flow cytometry using PE-Vio770-BDCA2 (Miltenyi Biotec), ECD-CD123 (Beckman Coulter, Villepinte, France), was >90%.

PBMCs were incubated with increasing doses of As₂O₃, activated with CpG-A for 6 h, and 1 μL/mL Golgi plug (BD Biosciences) was added in the last 3 h. The cells were firstly stained with FVD eFluor™ 506, then with PE-Vio770-BDCA2 (Miltenyi Biotec) and ECD-CD123 (Beckman Coulter). Cells were then stained with FITC-IFN-α and APC-Vio770-TNF-α (both from Miltenyi Biotec) with Cytofix/Cytoperm Buffer (BD Biosciences).