Flow cytometry analysis (FACS)

Cells and MVs were analyzed by flow cytometry using a BD FACSCanto II and the BD FACSDiva software to acquire data. For cell analysis, A549 stained with MitoTracker dyes and challenged with PLY as described above, were collected by trypsinization, washed, resuspended in HBSS, and analyzed by FACS. For MV analysis, forward scatter (FSC) and side scatter (SSC) were set at logarithmic gain. No threshold was applied. To set the MV gate (events less than 1 μm), we used 1 μm sizing calibration beads (Sigma-Aldrich). BD CS&T beads consisting of 3-μm bright beads, 3-μm mid, and 2-μm dim polystyrene beads were also run in parallel. In addition, to confirm the gating strategy, fresh human platelets, which are known to have a size of 2–4 μm, were analyzed.

For MV staining, all antibodies/dyes used were from Biolegend unless specified. MVs derived from A549 were stained with annexin V-APC in HBSS+/+(with Ca^2 + /Mg^2 +). As a negative control, MVs were stained with annexin V-APC in PBS containing 5 mM EDTA (annexin V binding to MVs requires calcium, which is removed by the addition of EDTA). To confirm the vesicular nature of the isolated vesicles, MVs were treated with Triton X-100 and analyzed by FACS. A549-MVs were also analyzed after staining with CFSE (Carboxyfluoresceinsuccinimidyl ester) and CellMask Deep Red plasma membrane stain (ThermoFisher). For this, MVs were incubated at 37 °C with CFSE or CellMask dyes for 20 min, and washed in PBS before analysis. CSFE and CellMask solutions were centrifuged at high-speed for 5 min before added to MVs. As a negative control, samples (PBS) that contain the dyes but not MVs were processed in parallel. For MVs from MitoTracker-labeled cells, MVs were isolated, resuspended in PBS and analyzed by FACS. MVs from human lung tissue or in BALF were analyzed after double staining with annexin V and anti-human or anti-mouse EPCAM-Alexa 488, respectively, in HBSS+/+. As a negative control, MVs were stained with annexin V-APC in PBS containing 5 mM EDTA and the corresponding isotype control antibody. All buffers used for MV analysis were double filtered (0.22 μm). For MV quantification, 5 μl of CountBright absolute counting beads (ThermoFisher) were added in each tube before analysis. To assess background signals, several negative controls were used (a) buffers with antibodies alone were analyzed to evaluate the background fluorescence of antibodies, (b) plain media after staining with antibodies to detect artifacts in the media, (c) single-stained MVs, (d) buffers containing only CFSE, CellMask, or MitoTracker dyes. FACS data were analyzed using the FCS Express 6 Flow cytometry software (Denovo Software, Glendale, CA).