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. 2023 Aug 3;26(9):107526. doi: 10.1016/j.isci.2023.107526

Figure 1.

Figure 1

Multiple extracellular vesicle populations produced by WEHI-231 cells contain immunoglobulin M (IgM)

(A) WEHI-231 cells were imaged by bright field microscopy at the time of plating (day 0, left) and after 24 h (day 1, right) in complete media. Cell clusters, interspersed with single cells, are indicated with a turquoise arrowhead.

(B) WEHI-231 cells were evaluated by flow cytometry for expression of IgM, IgD, CD19, CD5, CD23, and CD43 (blue) compared to fluorescence minus one (FMO) stained control cells (gray). Data are representative of two independent experiments.

(C) IgM in 2000 × g supernatant (2kG supernatant) from untreated or LPS (10 μg/mL) treated WEHI-231 cells at day 2 (columns 1,2) and at day 3 (columns 3,4) was determined by anti-mouse IgM ELISA. Data are from one (day 2) and two (day 3) independent experiments. Data are represented as mean ± SEM.

(D) Conditioned media from untreated WEHI-231 cells, or WEHI-231 cells treated with 1 or 10 μg/mL LPS, was centrifuged at 2000 × g and IgM expression in the resulting supernatant (8 μL/well) was determined by SDS/PAGE and immunoblot. Presented are immunoblots for IgM in samples prepared with unreduced (left, upper) or reduced (left, lower) sample buffer. Right image displays a longer membrane exposure of the unreduced samples, with low molecular weight IgM indicated by turquoise arrowhead. Purified mouse IgM (25 ng) was run as a positive control.

(E) Lysate preparations of whole cells, and extracellular vesicle (EV) populations isolated by differential ultracentrifugation (2 kG, 11,000 × g (11 kG), 110,000 × g (110kG)), from untreated or LPS (10 μg/mL) treated WEHI-231 cells were evaluated for IgM by unreduced immunoblot. Total protein was 1 μg for all samples. Purified mouse IgM (3.1 ng) was run as a positive control in a non-contiguous lane and is presented at the far right in both images.

(F) IgM in 11 kG EV (left), 110 kG EV (center), and 110kG supernatant (right) was determined by ELISA under buffer only or buffer +0.25% TX100 conditions, as indicated.

Data in (D) and (E) are representative of at least two independent experiments. See also Figure S1.