Anti cd86 percp efluor 710

For detachment, adherent macrophages were washed with PBS, incubated with 300 µl 10× Gibco™TrypLE™Select (Gibco, Life Technologies) at 37 °C for 15 min and gently collected with a cell scraper after adding 300 µl PBS. Before staining, Fcγ receptor blockage was performed by incubating the cells with 3 mg/ml Beriglobin P (#I4506, Sigma Aldrich) at 4 °C in the dark for 30 min. To determine the purity of isolated primary CD14+ monocytes, cells were stained with either FITC-conjugated mouse anti-human CD14+ REAfinity (#130-110-518, Miltenyi Biotec), FITC-conjugated human IgG1 REAfinity control (#130-113-437, Miltenyi Biotec), PE-conjugated mouse anti-human CD19 monoclonal (clone LT19, #130-091-247, Miltenyi Biotec), PE-conjugated monoclonal isotype control (#120-002-723, Miltenyi Biotec), FITC-conjugated mouse anti-human CD3 monoclonal (#130-080-401, Miltenyi Biotec) and FITC-conjugated mouse IgG2a isotype control (#130-091-837, Miltenyi Biotec) antibodies. To determine the frequency of CD86+ macrophages, cells were stained using anti-CD86-PerCP-eFluor 710 (eBioscience) or isotype control (IgG2b-PerCP-eFluor-710, eBioscience) antibodies. Data were acquired using a BD LSRFortessa™ flow cytometer and analysed using FlowJo software, Treestar Inc.

C57BL/6 (H-2^b) mice-derived MSCs were treated without (control) or with 10% LICM for 3 days; then the cells were blocked with 10% rat serum and incubated with anti-CD80 PerCP-eFluor 710 (eBioscience, cat. no. 46080180), anti-CD86 PerCP-eFluor 710 (eBioscience, cat. no. 46086280), anti-PD-L1 PerCP-eFluor 710 (eBioscience, cat. no. 46598280) and anti-Fas APC (eBioscience, cat. no. 17095180) mAbs. Flow cytometry was performed with BD FACSJazz; and data were analyzed with FlowJo software.