Figure 2.
Monomeric IgM is enriched in WEHI-231 EV and is detected at a greater level following detergent solubilization
(A) Equivalent amounts of 11kG (upper row) and 110kG (lower row) EVs isolated by differential ultracentrifugation were titrated and adsorbed onto aldehyde sulfate microbeads, stained for surface expression of IgM (or isotype control), canonical EV marker proteins, and negative control protein (CD4), and analyzed by flow cytometry. Beads incubated with buffer alone were included as a control. Histogram overlays display the fluorescence for each antigen (CD4, CD9, CD63, CD81, or IgM). The graph indicates the percentage of beads positive for IgM compared to beads stained with isotype control antibody. Data are representative of at least three independent experiments.
(B) WEHI 231 cells (16 × 10 cm plates; 0.1 × 106 cells/mL media) were cultured for two days, after which EV were isolated by DUC, resuspended in 1.1 mL PBS and serially titrated at 1:10, and subsequently analyzed by nanoparticle tracking analysis. Displayed are the starting and ending cell number (left) and cell viability (right).
(C) Histogram plots for particle diameters of 11kG (left) and 110kG (right) samples are presented.
(D) Displayed are the particle concentration (left), mode diameter (center), and percentile distribution by particle size (right) of 11kG and 110kG samples. N = 2 independent experiments.
(E) Whole cell and isolated EV (2kG, 11kG, 110kG) lysates (2.3 μg protein/well) were evaluated for IgM and CD9 expression by SDS/PAGE and immunoblot. Total protein (left) and immunoblot detection of IgM and CD9 under reduced (right, top) or unreduced (right, bottom) conditions are presented. A longer (upper) and shorter (lower) exposure are presented for the unreduced immunoblot. Turquoise arrowheads (total protein gel) indicate visible discrepancies in expression of protein species across the four lysate preparations. Purified IgM (2.5 ng) and 110kG supernatant (10.5 μL) were run as control samples. N = 2 independent experiments.
(F) The amount of IgM in 11kG and 110 kG EV populations under buffer only (no TX100) and buffer +0.25% TX100 conditions (top graph) and the total IgM in 11kG plus 110 kG EV populations combined as compared to 110kG supernatant (bottom graph), determined by anti-mouse IgM ELISA, are presented.
Data are representative of three independent experiments. ANOVA and Tukey’s post-hoc analysis was applied to data presented in (A) and (F) prior to direct comparison. ∗ = p < 0.05; ∗∗ = p < 0.005; ∗∗∗ = p < 0.001. Data are represented as mean ± SEM. See also Figures S2 and S3.