In Vivo Model of Peritonitis Induced by Thioglycollate

Thioglycollate medium 3% (Sigma-Aldrich) was prepared in sterile conditions and protected from light at least 6 months before experiments. Mice were injected intraperitoneally (IP) with 2 mL of thioglycollate solution and euthanized on day 4. Mice treated with MVs-MSCs were injected IP with four doses of MVs-MSCs (8.38 × 10⁹ particles) at 0, 24, 48, and 72 h. In contrast, control mice were treated at the same time-points but using vehicle (PBS). Finally, on the 4th day after thioglycollate infusion, an peritoneal lavage was performed with 5 mL of cold sterile PBS. The peritoneal exudate was centrifuged at 300 g for 5 min and cells were processed by FACS and the supernatant was analyzed by MILLIPLEX (Merck Millipore, USA). During the standardization of the model of peritonitis, washes at 24, 48, 72, and 96 h were performed to identify the cell cluster with GR1, CD19, and CD11b phenotype by FACS (Figures S4A,B in Supplementary Material). Furthermore, the predominant presence of Mϕs in the last day of the induction of peritonitis was confirmed by morphological analysis (HE) and immunohistochemistry for CD11b (Figure S4C in Supplementary Material).