Fig. 4

EV detection in reduced volume of plasma. A Comparison of EV detection in decreasing volume. 3000 anti-CD63 beads were used per test in different incubation volumes (100 µl, 50 µl and 20 µl), using either 5 ml tubes or microtiter plates (96-well plates V-bottom, U-bottom or flat bottom, as indicated). Lung cancer-derived EVs (from H3122 cell line) were obtained by ultracentrifugation, incubated with beads for 16 h and, after staining with anti-CD81-PE, washed and analysed by flow cytometry. EV concentration was the same in all the conditions (4 × 10⁶ H3122 EVs/µl). Relative Fluorescence Intensity (RFI) obtained in each condition is represented. Three independent experiments are shown. Statistical analysis was performed by a Two-way ANOVA Fisher’s LSD test comparing each condition to the 100 µl tube condition as independent comparisons. (*p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001). B Detection of EV proteins directly in plasma. 12 µl of healthy donor plasma were incubated with 3000 anti-CD63, anti-EpCAM or IgG isotype control beads in a final volume of 25 µl. Captured EVs were detected with anti-CD81-PE. Left. Detection of EVs directly in 12 µl of healthy donor plasma. Right. 3.14 × 10⁸ H3122-derived EVs (from the H3122 cell line) were added to 12 µl of the same healthy donor plasma (2.6 × 10⁷ H3122 EVs/µl plasma). C EpCAM and MICA, can be detected directly in minimal volumes of plasma from a cohort of lung cancer (LC) patients by flow cytometry. 12 µl of PBS-1% casein containing 3000 beads conjugated either with anti-CD63, anti-EpCAM or anti-MICA were incubated for 16 h with 12 µl of plasma (EDTA-tubes) from a cohort of lung cancer patients (12 initial stage and 12 advanced stage) and 12 healthy donors. The final volume of the assay was 24 µl. EVs captured in each assay were detected with anti-CD81-PE, or IgG-PE as a negative control to calculate the Relative Fluorescence Intensity (RFI). The mean RFI from three independent repetitions was calculated for each patient and represented as a violin plot for each group of patients. Statistical analysis was performed by one-way ANOVA (Tukey test for correction of multiple comparisons) or Krustal-Wallis non-parametric test (Dunns test for correction of multiple comparisons) with the same results. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Patient samples were obtained at Clínica Universidad de Navarra