Penicillin g streptomycin p s

Rat MSC were isolated form CIA and control femurs, as previously described [41 (link)]. Briefly, bone marrow was flushed with PBS and bone debris were removed by filtering the cell suspension through a 100 µm cell strainer. After red blood cells lysis, cells were counted and seeded in minimum essential medium alpha modification (α-MEM) supplemented with 10% MSC-qualified fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA MA, USA) and 1% penicillin G-streptomycin (P/S; Invitrogen, Carlsbad, CA, USA), at 10⁶/100 mm plate. MSC were selected by adherence to plastic surface, expanded and cryopreserved for forward use. Isolated and cultured MSC identity was confirmed by multicolor flow cytometry based on classical cell surface markers CD29⁺ (anti-CD29-APC, HMβ1-1, BioLegend, San Diego, CA, USA), CD90⁺ (anti-CD90-PE, MRC OX-7, Immunotools, Friesoythe, Germany) and CD45⁻ (anti-CD45-FITC, MRC OX-30, Immunotools, Friesoythe, Germany) staining, and by ability of cells to differentiate into the osteogenic, chondrogenic and adipogenic lineages. Cells from CIA and control animals were maintained in a humidified incubator, at 37 °C and 5% CO2, and used for experiments between passages 4 and 8.

Monocytes were isolated from buffy coats of healthy blood donors using RosetteSep™ human monocyte enrichment antibody cocktail (STEMCELL Technologies), as previously described⁵³ . DC cultures were set up with 60–100 × 10⁶ human peripheral blood monocytes, plated at 20 × 10⁶ cells per 100 mm diameter petri dishes, in DC differentiating media - RPMI-1640 + Glutamax (Invitrogen) cell culture media, with 10% heat inactivated FBS (Lonza) and 1% penicillin G-streptomycin (P/S; Invitrogen), further supplemented with 50 ng/mL IL-4 and GM-CSF (Immunotools). After 4 days of differentiation, cells were washed with PBS and re-cultured for 3 additional days in EV producing media. This media was obtained from ultracentrifuged (100,000 × g, for 2 h at 4 °C) RPMI supplemented with 10% FBS, which was then diluted to 1% FBS, and further supplemented with 1% P/S, IL-4 and GM-CSF, as above. Absence of bovine-EV in the ultracentrifuged RPMI + 10% FBS media was confirmed by transmission electron microscopy (Supplementary Fig. S3). Cells were maintained in a humidified incubator, at 37 °C and 5% CO₂. DC cultures in EV-producing media were monitored by optical microscopy and no changes in cell morphology or general culture condition were observed (Supplementary Fig. S4).