Figure 3.

WEHI-231 11kG and 110 kG EV populations fractionated by sucrose gradient centrifugation reveal correlated expression of IgM and CD81

EV were first isolated by differential ultracentrifugation and then subjected to density gradient fractionation by overlay of sucrose/PBS layers with decreasing density. Fractions were collected (F1 least dense to F6 most dense) and washed with buffer. An equivalent proportion of each resuspended fraction was subsequently utilized for downstream analyses.

(A) Density gradient fractions of EV were subjected to PAGE and immunoblotted for IgM and CD81, as indicated. Purified mouse IgM was run as a positive control. The measured density (g/mL) of each fraction are shown (right graph). N = 2 independent experiments.

(B) Density gradient fractions of EV were tested by anti-mouse IgM ELISA under buffer +0.25% TX100 conditions for 11kG (left) and 110kG (right) EV populations.

(C) Density gradient fractions were adsorbed onto aldehyde sulfate microbeads, stained for IgM and CD81, and analyzed by flow cytometry. Dot plots for IgM (top), CD81 (middle), and isotype control (bottom) of fraction 3 are shown, with gates indicating the positive bead populations. The graphs (bottom) show the percentage of positive beads for all 11 kG EV (left) and 110 kG EV (right) fractions.

(D) Biparametric dot plots of buffer (left), fraction 3 11 kG EV (center), and fraction 3 110 kG EV (right) samples displaying the expression of isotype control/CD81 or IgM/CD81 are presented.

Data presented in (A)–(D) are representative of at least two independent experiments. Data are represented as mean ± SEM.