Figure 5.

EV isolated from WT and μsKO mouse peritoneal cavity fluid and peritoneal B cell cultures contain low molecular weight IgM

(A) Peritoneal cavity washout fluid was obtained from WT and μsKO mice and subjected to DUC to isolate 2kG, 11kG, and 110 kG EV populations, which were then resuspended in an equivalent volume of buffer, resolved by SDS/PAGE under unreduced conditions, and immunoblotted for IgM and CD81, along with 2kG and 110kG supernatant samples. Data are representative of two independent experiments.

(B) IgM expression in the peritoneal cavity samples described in (A) was determined by anti-mouse IgM ELISA under buffer +0.25% TX100 conditions. Results are shown for the EV samples (left) and 110kG supernatant (right).

(C) Peritoneal B-1a cells (CD5⁺B220^loCD43⁺CD23⁻CD19⁺), peritoneal B2 (B2P) cells (CD5^-B220^highCD43⁻CD23⁺CD19⁺), and splenic B2 (B2 S) cells (CD5^-B220^highCD43⁻CD23^highCD21^lo/−CD19⁺) were sorted from WT and μsKO, as detailed in materials and methods. The sorted B-1a, B2P, and B2 S cells were cultured in media alone or media + LPS (25 μg/mL) for 24-h, after which conditioned media was harvested and the 11kG and 110 kG EV populations were subsequently isolated by differential ultracentrifugation. EV samples (11 kG EV, left and 110 kG EV, right) were resuspended in an equivalent volume of buffer, resolved by SDS/PAGE under non-reducing conditions, and immunoblotted for IgM.

Data are representative of at least two independent experiments. See also Figures S5 and S6.