Ls magnetic column

Single-cell suspensions of splenocytes were obtained from naive, infected/non-treated and infected/treated mice at 8 and 14 days p.i. Spleen cell suspensions were obtained by mechanical dissociation in PBS, then filtered in 0,70 µm strainer. 20% of each spleen was used for immunophenotyping by FACS, 80% left was dedicated to neutrophils and monocytes sorting. Red blood cells were lysed (ACK, Lonza) and an enrichment with biotinylated anti-B220 (BD Biosciences), anti-CD3 (BD Biosciences), following of anti-biotin Ab magnet-bead coupled (Miltenyi) and magnetic LS-columns (Miltenyi) was performed to remove spleen lymphocytes and increase the sorting efficacy. Cells were stained with specific markers of populations of interest (Ly6G-BD BioSciences, Ly6C-BioLegend, CD11b-BD BioSciences) and neutrophils (Ly6G^hi) and inflammatory monocytes (Ly6C^hi) were sorted using the BD Biosciences FACSAria device. Sorted cells (>97-98% pure) from naive, infected/non-treated and infected/treated mice showed a viability > 90%. They were cultured in U bottom 96-well plates at a density of 4 × 10⁶ cells/ml (1 × 10⁶ cells/250 µl/well) in 10% FBS-containing RPMI medium. After 24 h of cell culture, cell-free supernatants were collected and stored at −20°C, to allow cytokines and chemokines protein release quantification.

Cell types were isolated from WAT and BAT as described previously (46 (link)). In brief, inguinal depots of WAT or interscapular depots of BAT of 4 mice were pooled and digested for 45 min using a collagenase D (1.5 U/ml) and dispase II (2.4 U/ml) containing buffer. After filtering through a 100 µm cell strainer, the flow through was pelleted by centrifugation for 5 min, at 600 x g. The remaining sample was resuspended (PBS containing 2 mM EDTA, 0.5% BSA, 2 mM glucose), passed through a 40 µm cell strainer and centrifuged (5 min, 600 x g). The cell pellet was resuspended and incubated for 15 min with CD11b MicroBeads (Miltenyi, 10 µl beads/10⁷ cells) for isolation of the macrophage fraction. The CD11b+-fraction was separated using magnetic LS-columns (Miltenyi). The remaining sample was incubated with CD31 MicroBeads (Miltenyi) for endothelial cell isolation. The remaining flow through fraction was considered as adipocyte pool. Cell fractions were centrifuged and the obtained pellets dissolved for RNA isolation in TRIzol^® reagent.

Bone marrow was harvested from male transgenic mice 20 weeks post tamoxifen administration. Bone marrow cells were incubated with ACK red cell lysis buffer to remove erythrocytes and depletion of lineage positive cells was performed using the MACS Lineage Cell Depletion kit for mouse (Miltenyi Biotech, Cat#130–090–858) and magnetic LS columns (Miltenyi Biotech, Cat#130–042–401) according to the manufacturer’s instructions. Lineage depleted cells were stained with fluorophore-conjugated antibodies targeted against cell surface markers (Supplementary Table 1) as above and 4′,6-Diamidino-2-Phenylindole (DAPI) was used as a viability marker. Viable KL (cKit⁺Sca1⁻Lin⁻) were sorted on BD FACSAria Fusion 5, BD FACSAria II and BD FACSAria Fusion 3 (BD Biosciences) and used for transplantation and molecular analyses.

Femurs from 7 weeks old WT mice were collected and rapidly flushed in sterile conditions with RPMI/10% FBS and smashed through a 100 μm filter to remove aggregates and centrifuge at 1,400 rpm for 5 min. The pellet was resuspended in PharmLyse solution (BD Biosciences) to lyse red blood cells. Lysis reaction was stopped by adding FBS and then centrifuged 5 min at 1,400 rpm. Pellet was resuspended in PBS-FBS 0.5%-EDTA 2 mM buffer prior to isolation. Neutrophils were isolated using the Neutrophil Isolation Kit (MiltenyiBiotec) following manufacturer instructions. Briefly, cells were stained with a cocktail of biotin-conjugated monoclonal antibodies not expressed on neutrophils. After that, cells were stained with magnetic anti-biotin antibodies and separated using magnetic LS Columns (MiltenyiBiotec). Cells unretained by the colums were numerated and seeded in a 96 well-plate at 1.10⁵ cells per well prior to stimulation. Supernatant was collected after 4 h stimulation and stored at −80°C for further analysis. Cell death was monitored by MTT using a standard protocol. Thiazollyl blue tetrazolium bromide (Sigma) solution was added onto the cells after supernatant collection and incubated for 2 h at 37°C, and a 10% SDS acetic acid solution was then added. MTT reduction to formazan was quantified by an absorbance microplate reader (EL800, BioTek, Colmar) at 610 nm (KC4 software).

CD31⁺ cells were isolated using magnetic LS columns (Miltenyi Biotec) and flow cytometric analysis was conducted as previously described 3 (link),11 (link). Antibody used included the following: CD3, CD11b, CD14, CD19, CD31, CD34, CD45, CD146 (BD Bioscience, San Jose, CA, USA), CD133 (AC133; Miltenyi Biotec) and KDR (R&D Systems, Minneapolis, MN, USA). Flow cytometric data were analysed with FlowJo (Tree Star, Inc., Ashland, OR, USA) using appropriate isotype control.

PBMCs were collected from whole blood of healthy donors. PBMCs were obtained from buffy coats by Ficoll-Paque (GE Healthcare Biosciences, Piscataway, NJ; catalog 17–1440-02) density gradient centrifugation at 400 g at 20 °C for 40 min to separate blood constituent parts. After purified cells were washed with PBS, CD14+ monocytes were isolated using the Pan Monocyte Isolation kit (Miltenyl Biotec, San Diego, CA; catalog 130–096-537) followed by separation using magnetic LS columns (Miltenyl Biotec; catalog 130–042-401), according to the manufacturer’s protocol.
Macrophages were differentiated from these monocytes according to the previous reports [31 (link), 32 (link)]. The monocytes were cultured in RPMI1640 with either GM-CSF at 20 ng/ml (named M0-GM) or M-CSF at 20 ng/ml (named M0-M) for 5 days. M0-GM macrophages were then stimulated by LPS (20 ng / ml) and IFN-γ (20 ng / ml) for 4 days to polarize into CD80+ / CD86+ (M1) macrophages. On the other hand, M0-M macrophages were stimulated by IL-4 (20 ng / ml) and IL-13 (20 ng / ml) for 4 days to polarize into CD163+ or CD206+ (M2) macrophages (Fig. S3A-C). M0-GM or M0-M macrophages were stimulated by EVs (20 μg / ml) for 4 days to assess the effects of EVs.