Counting beads

Mouse lungs were minced to single cell suspensions and filtered through 70‐μm cell strainers (BD Biosciences, San Jose, CA) as previously described.9, 28 The erythrocytes were lysed using red blood cell lysis buffer (BD Biosciences) and washed with phosphate‐buffered saline. Cells were labeled with monoclonal antibodies including anti‐mouse CD45‐FITC, Gr‐1‐PE, CD11b‐PerCP, CD48‐APC, and counting beads (BD Biosciences) was added. Matched isotype antibodies were used as negative controls. All samples were analyzed using a FACSCalibur cytometer (BD Biosciences) and CellQuest software, and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

EMVs were immunophenotypically defined as CD45−/CD42b−/CD31 + (31). Blood samples were pre-diluted 1:100 in buffered saline solution (PBS) and 50 μL of the diluted solution were used for cell-labeling reaction. Anti-human monoclonal antibodies with suitable volumes were added to the diluted sample: 5 μL of FITC CD45 (BD, clone HI30), 2 μL of CD31 Alexa Fluor 647 (BD, clone WM59) and 5 μL of CD42b PE (BD, clone HIP1). The samples containing monoclonal antibodies were incubated for 30 min protected from light at room temperature, and 2 mL of PBS was added; they were then centrifuged for 5 min at 540 g. The supernatant was discarded and the pellet resuspended with 500 μL PBS and 50 μL counting beads (BD Biosciences, San Jose, California) were added. A total of one million events were acquired using a flow cytometer (FACSCanto II) (BD Biosciences, San Jose, California) and analyzed using by Infinicyt version 1.7 (Cytognos, Salamanca, Spain). A blinded investigator processed all samples. The amount of EMVs was determined from the number and volume of counting beads. Microvesicle sizes were compared to platelet sizes using average fluorescence intensity of forward scatter, showing a mean diameter ≤ 1.0 μm. Figure S5 (supplementary material) describes the gating strategy for CD45−/CD42b−/CD31 + EMVs. EMV was assessed 10 min before and 10 and 70 min post-intervention.

T cells were stained for CD3-PB (Pacific Blue) and sorted with an Influx apparatus (BD), while flow cytometric analyses were performed with Influx or Gallios (Beckman Coulter, Brea, CA, USA) flow cytometers. Data were analyzed with FlowJo 10.6.2 software (Treestar, Woodburn, OR, USA). Additionally, we used counting beads (BD Biosciences, 340334, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions to determine the % of Nes-GFP⁺ cells. The primary and secondary antibodies are listed in the Supplementary Information, Table S2.

Single-cell suspensions were isolated from freshly harvested murine livers by mechanical and enzymatic digestion as previously described [6 (link)]. For flow cytometry analysis, cell suspensions were stained with fluorochrome-conjugated antibodies for CD45, Ly6G, CD103, Ly6C, B220, NK1.1, Counting beads (BD Bioscience, Heidelberg, Germany), CD11b, F4/80, CD11c, MHCII, CD3, CD4, CD8, CD25, CD62L, FoxP3, viability dye (eBioscience, San Diego, CA, USA), α-GalCer (NIH) and analyzed using a LSR Fortessa flow cytometer system (BD Bioscience, Heidelberg, Germany). NKT cells were gated as singlets, viable, CD45+, CD3+ and NK1.1+ signals. For detailed gating strategy of all immune subsets see Supplementary Table S2. Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).