Cytometer setup tracking module

Tear samples were analysed as previously reported^{16 (link),19 (link),20 (link),35 (link)}. Briefly, 100 μL of tears were stained using a reagent mix, as detailed in Table S1. Samples were then incubated for 45 min, at room temperature, in the dark. After adding 200 μL of PBS 1X to each tube, 1 × 10⁶ events/sample were acquired by a FACSVerse flow cytometer (BD Biosciences). The trigger threshold was set on the allophycocyanin (APC) channel, which is the channel in which the LCD (a pan EV marker) emits (threshold placed at 200/262,144)^{20 (link),36 (link)}. For all used parameters the height (H) signals and the bi-exponential/logarithmic modes were selected. Current guidelines for flow cytometry analyses and for EV studies were taken into account^{37 (link),38 (link)}. The Cytometer Setup & Tracking Module (BD Biosciences) was used for the daily quality check. Compensation was assessed using CompBeads (BD Biosciences) and single stained fluorescent samples. Data were analysed using FACSuite v 1.0.6.5230 (BD Biosciences) and FlowJo X v 10.0.7 (TreeStar, Ashland, OR, USA) software. EVs concentrations were obtained by the volumetric count function. With the used dilution no swarm effects occurred.

Samples were acquired by using a FACSVerse flow cytometer (BD Biosciences - three laser, eight colour configuration). Data reproducibility and flow cytometer performances were monitored by the Cytometer Setup & Tracking Module (BD Biosciences). Compensation were assessed using CompBeads (BD) and single stained fluorescent samples. Data were analyzed using FACSDiva v 6.1.3 (BD), FACSuite v 1.0.5 (BD) and FlowJo v 8.8.6 (TreeStar, Ashland, OR, USA) software.

Water samples collected at 1, 24 and 78 m were treated with 1% Triton X-100 (30 min) in order to remove any biological particles. Samples were then acquired by flow cytometry (FASCVerse, BD Biosciences, San Jose, CA, USA). To avoid swarm effects, sample dilution was established as recommended [29 (link)]. In any case, the flow rate resulted in event counts ≤2000 events/second. Diluted samples were then acquired and 1 × 10⁵ events/sample were registered. Side Scatter and Forward Scatter (FSC) MegaMix-Plus beads (Byocitex, Marseille, France) were used to measure the particle size [30 (link),31 (link)]. MegaMix-Plus SSC and FSC are mixes of fluorescent polystyrene beads of known diameters selected to cover the size range 0.1–1 μm (SSC beads: 0.16, 0.20, 0.24 and 0.50 μm; FSC beads: 0.1, 0.3, 0.5 and 0.9 μm). By acquiring the beads using the same setting applied for sample analysis, it is possible to identify, in the scatter plots, the particle diameter ranges, given that the scattered light (FSC and/or SSC) is proportional to the particle diameter [32 (link)]. The same parameters were applied for all other analyses. Instrument performances, data reproducibility and fluorescence calibrations were sustained by the Cytometer Setup & Tracking Module (BD Biosciences) [32 (link)].