Fig. 5

Double antibody staining of IP complexes allows for meaningful quantification of surface markers on plasma-recovered EVs. (A) Platelet EVs isolated from plasma with anti-CD61 beads were stained with CD41-PE and CD9-AF488 in single or double staining settings. Fluorescence signal for both markers was equivalent, despite the incubation with 1 or 2 antibodies simultaneously. (B) Triple-coated or anti-CD61 beads were incubated in untreated, thrombin treated or 56 °C heated plasma and double stained with CD41-PE and CD9-AF488. S/N ratios obtained with CD9-AF488 were plotted. A significant drop in fluorescent signal could be appreciated only upon 56 °C treatment, when CD61 beads were employed. (C) Measurements of CD41-PE, referring to the experiment described in (B). Substantial losses in CD41-PE signal were detected after staining IP complexes recovered with both beads, across treatments. Untreated and thrombin from triple-coated and CD61 beads, respectively, are shown as duplicates