Figure 7.

IgM contained in B cell-derived EV can accumulate intracellularly in secondary cells

EV (11kG and 110kG) were isolated from WEHI-231 cell culture supernatant and subsequently applied to HeLa cell monolayers, followed by HeLa cell lysate preparation and immunoblot analysis or immunofluorescent confocal microscopy of intact monolayers.

(A) Schematic showing the experimental procedure, and designation of buffer, 11 kG EV, or 110kG (EV) as (input) and HeLa lysates as (output).

(B) Immunoblot for IgM and CD81 in isolated EV prior to application (left, (input)) and in HeLa lysate preparations generated 24 h post-EV application (right, (output)). Mouse IgM was run as a positive control. Data are representative of two independent experiments.

(C) Representative confocal microscopy images of HeLa monolayers 1 h (left set) and 4 h (center set) after application of 11 kG EV. Images show IgM alone (anti-mouse IgM AF488), and pseudocolored IgM (green) with actin (phalloidin rhodamine, red) and nuclei (dapi, blue). HeLa monolayers 4 h after application of vehicle (PBS) (right set) that were subjected to the identical staining reagents utilized above are shown.

(D) Representative microscopy image of one confocal layer (0.75 μm) 24 h post-application of 11 kG EV (left set). A confocal microscopy image of HeLa treated with buffer only, that were subjected to the identical staining reagents utilized above, is presented (right set). A region of interest (yellow box) from five sequential layers of HeLa treated with the 11 kG EV was magnified and is presented in the lower panel (1–5).

Data in (C) and (D) are representative of two independent experiments. Scale bar indicates 20 μm. See also Figures S7–S9.