Immunomodulatory effects of dental pulp stem cells on lymphocytes and monocytes from patients with rheumatoid arthritis Part 2

.FCS are flow cytometry files Immune checkpoint expression was evaluated on DPSCs DPSCs were detached with trypsin/EDTA (Gibco, ThermoFisher) and analyzed by flow cytometry to quantify the expression of PD-L1, PD-L2, CD155, Galectin-9, CD80, CD86, and 4-1BBL. Cells were suspended in 100 μL PBS with 0.1% Fixable Dead Cell Stain near-IR-fluorescent reactive dye (Thermo Fisher Scientific), and stained for 15 min at room temperature. After washing with PBS + 1% FBS, cells were suspended in 100 μL PBS + 1% FBS containing the following antibodies combined in two mixes: (1) PerCP anti-human CD45 (clone REA747), PE anti-human CD155 (clone REA1081), PE-Cy7 anti-human PD-L1 (clone 29E.2A3), APC anti-human CD80 (clone REA661) and FITC anti-human CD86 (clone REA968); (2) PerCP anti-human CD45 (clone REA747), PE anti-human 4-1BBL (CD137L) (clone REA254) and APC anti-human PD-L2 (clone REA 985). Cells were stained for 30 min on ice. After washing, cells stained with mix 1 were suspended in 300 μL PBS + 1% FBS for subsequent acquisition while cells stained with mix 2 were fixed and permeabilized for 20 minutes at 4°C using 250 µl BD fixation/permeabilization solution (BD Biosciences). After washing twice with 1 ml BD permeabilization/washing buffer (BD Biosciences), cells were stained with the FITC anti-human Galectin 9 antibodies (clone 435) in 100 µl BD permeabilization/washing buffer for 30 minutes at 4°C. After washing with 1 ml BD permeabilization/washing buffer, cells were suspended in 300 μL PBS + 1% FBS to be acquired. All cells were acquired with the FACS Canto II flow cytometer (BD Biosciences) equipped with two lasers for excitation at 488 and 633 nm. All antibodies were purchased from Miltenyi Biotec, except PE-Cy7 anti-human PD-L1 which was purchased from BioLegend Inc, and used as suggested by the manufacturers. Labeling of the FCS files. Files indicate: Experiment number _ Patient number _ Tube number _ Sample type and stimulus _ Mix number Excel files report the IL-6 ELISA results and the Luminex Assay results The files report sample names (Experiment number _ Patient number _ Sample type and stimulus) and the concentrations of the cytokines in pg/ml. <LLD indicate values lower than the lower limits of detection.