*2.4. Flow Cytometry*

Cell suspensions from PECs, livers, and spleens were washed twice with PBS containing 2% FBS and 0.1% sodium azide (FACS buffer), stained with specific antibodies for 30 min at 4 ◦C as previously published [31]. The following antibodies were used: anti-Sirp*α* (P-84), -CD11c (N418), -CD86 (GL1), CD8 (53-6.7), -Siglec-F (E50-2440), -F4/80 (BM8), -CD11b (M1/70), -CD40 (HM40-3), -CD80 (16-10A1), and I-A/I-E (M5/114.15.2). Expression of FoxP3, HO-1, and IL-10 was analyzed by intracellular staining. Cells in which IL-10 was analyzed were incubated with Brefeldin-A for 6 h at 37 ◦C and phorbol myristate acetate (PMA, 200 nM) (Merk, Sigma-Aldrich, USA). After two washes with FACS buffer, cells were incubated with the following antibodies: anti-CD3 (17A2), -CD4 (RM4-5), -CD8 (53-6.7), or -F4/80 (BM8). After permeabilization with Cytofix and Perm wash buffers (Biolegend, USA), cells were incubated with IL-10 (JES5-1E3), FoxP3 (MF14), and HO-1 (clone ab13248 from Abcam, Waltham, MA, USA) specific antibodies. ROS/RNS produced by F4/80<sup>+</sup> cells were determined with 2 ,7 -dichlorofluorescein diacetate (DCFDA, Merk, Kenilworth, NJ, USA) probe, a fluorogenic dye that is oxidized into the fluorescent 2 ,7 -dichlorofluorescein. Briefly, cells were incubated in PBS for 30 min at 37 ◦C with DCFDA, washed with FACS buffer, and fluorescence was measured in a flow cytometer. Analyses were performed using a BD Accuri C6 Plus cytometer and software (BD-Biosciences). Antibodies were obtained from Biolegend (USA). Analyses were performed with Accuri C6 Plus software.
