Anti hla dr

Cell surface staining was performed using monoclonal antibodies fluorescently labeled with fluorescein isothiocyanate (FITC), allophycocyanin (APC) or phycoerythrin (PE). Anti-CD3 and anti-CD14 antibodies (ImmunoTools GmbH, Friesoythe, Germany) were used to stain T cells and monocytes, respectively. Anti-CD86 antibody (ImmunoTools), and anti-HLA-DR (Immunostep; Salamanca, Spain) were used to assess MoDC maturation. Data were acquired with Attune® Acoustic Focusing Cytometer and analyzed using Attune® Cytometric Software v2.1 (Applied Biosystems, Carlsbad, CA, USA. In each case, at least 10,000 events were acquired in the gate of interest. The forward scattered (FSC) and the side scattered (SSC) light were used to evaluate relative cell size and granularity and cell doublets were excluded based on FSC-A/FSC-H. Mean Fluorescence Intensity (MFI) was determined for each marker.

Roswell Park Memorial Institute (RPMI) medium (Invitrogen) was used for the cell cultures. The following antibodies were used: anti-CD14, anti-HLA-DR, anti-CD3 (Immunostep), and anti-PD-L1 (Miltenyi Biotec). The LPS from Salmonella abortus was a kind gift from Dr. Galanos (Max Planck Institute of Immunobiology and Epigenetics). Carboxyfluorescein succinimidyl ester (CFSE) for the proliferation assays was purchased from Thermo Fisher. The lymphocyte stimulus pokeweed (PWD) was purchased from Sigma-Aldrich. To inhibit PD-1/PD-L1 interaction, a fully human IgG4 (S228P) anti-PD-1 receptor-blocking monoclonal antibody was used (Bristol-Myers Squibb). All the reagents used for cell cultures were endotoxin-free, as assayed with the Limulus amebocyte lysate test (Cambrex).