Bead-based Multiplex EV Analysis by Flow Cytometry

Different sample types were subjected to bead-based multiplex EV analysis by flow cytometry (MACSPlex Exosome Kit, human, Miltenyi Biotec) (9 (link), 10 (link)), with details regarding sample preparation and normalization summarized in Table S2 in Supplementary Material. Unless indicated otherwise, EV-containing samples were processed as follows: Samples were diluted with MACSPlex buffer (MPB) to, or used undiluted at, a final volume of 120 µL and loaded onto wells of a pre-wet and drained MACSPlex 96-well 0.22 µm filter plate before 15 µL of MACSPlex Exosome Capture Beads (containing 39 different antibody-coated bead subsets) were added to each well. Generally, particle counts quantified by NTA, and not protein amount, were used to estimate input EV amounts. Filter plates were then incubated on an orbital shaker overnight (14–16 h) at 450 rpm at room temperature protected from light. To wash the beads, 200 µL of MPB was added to each well and the filter plate was put on a vacuum manifold with vacuum applied (Sigma-Aldrich, Supelco PlatePrep; −100 mBar) until all wells were drained. For counterstaining of EVs bound by capture beads with detection antibodies, 135 µL of MPB and 5 µL of each APC-conjugated anti-CD9, anti-CD63, and anti-CD81 detection antibody were added to each well and plates were incubated on an orbital shaker at 450 rpm protected from light for 1 h at room temperature. Next, plates were washed by adding 200 µL MPB to each well followed by draining on a vacuum manifold. This was followed by another washing step with 200 µL of MPB, incubation on an orbital shaker at 450 rpm protected from light for 15 min at room temperature and draining all wells again on a vacuum manifold. Subsequently, 150 µL of MPB was added to each well, beads were resuspended by pipetting and transferred to V-bottom 96-well microtiter plate (Thermo Scientific). Flow cytometric analysis was performed, unless indicated otherwise, with a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec; see Table S1 in Supplementary Material for acquisition parameters) by using the built-in 96-well plate reader. All samples were automatically mixed immediately before 70–100 µL were loaded to and acquired by the instrument, resulting in approximately 7,000–12,000 single bead events being recorded per well. FlowJo software (v10, FlowJo LLC) was used to analyze flow cytometric data. Median fluorescence intensity (MFI) for all 39 capture bead subsets were background corrected by subtracting respective MFI values from matched non-EV buffer or media controls that were treated exactly like EV-containing samples (buffer/medium + capture beads + antibodies). GraphPadPrism 6 (GraphPadPrism Software, La Jolla, CA, USA) was used to analyze data and assemble figures. To generate heatmaps of data, flow cytometric data were gated in FlowJo with gated data being exported to comma separated files, which were subsequently imported into MATLAB (v9.3.0, Mathworks Inc.) for further analysis and data visualization. In order to compare data from the MACSQuant and FACS Symphony flow cytometers, the log10 transformed ratios of capture beads + EVs + Ab over their respective controls (capture beads + ab) was compared, rather than using background subtraction, which allowed for comparison despite axis scaling differences.