rat anti p28  (R&D Systems)

Journal: Frontiers in immunology

Article Title: IL-27-containing exosomes secreted by innate B-1a cells suppress and ameliorate uveitis.

doi: 10.3389/fimmu.2023.1071162

Figure Lengend Snippet: FIGURE 2 B-1a cells from the peritoneal cavity express membrane-bound IL-27. (A) B-1a cells from mouse peritoneal cavity were stimulated for 72 hours with anti-IgM/anti-CD40 and analyzed by intracellular cytokine staining assay. Graphs show absolute numbers of p28+, Ebi3+ or p28+Ebi3+ B cells in CD19+CD5+CD23- compartment (left panel) and IL-27-producing B-1a cells (right panel) (n=3/group). (B) B-1a cells were stimulated for 72 hours with anti-IgM/anti-CD40. The B-1a cells were stained with p28, Ebi3 or CD81 antibodies and FACS analysis was performed without permeabilizing the cells. Left panel shows absolute numbers of CD19+CD5+CD23- B-1a cells in the peritoneal cavity expressing IL-27 subunit proteins (p28, Ebi30 proteins). Right panel shows absolute numbers of B cells in CD19+CD5+CD23-CD81+ compartment that expressed p28 and/or Ebi3 on the B-1a plasma membrane(n=3/group). (C, D) Analysis and localization of cell surface expression of p28, Ebi3 or CD81 on activated i27-Breg cells by immunohistochemical staining and confocal microscopy (representative of three individual experiments).

Article Snippet: Cells were then incubated with rabbit anti-CD81 (Cell Signaling), mouse anti-Ebi3 and rat anti-p28 (R&D system either MAB7430 or MAB18342).

Techniques: Membrane, Staining, Expressing, Clinical Proteomics, Immunohistochemical staining, Confocal Microscopy

Journal: Frontiers in immunology

Article Title: IL-27-containing exosomes secreted by innate B-1a cells suppress and ameliorate uveitis.

doi: 10.3389/fimmu.2023.1071162

Figure Lengend Snippet: FIGURE 3 i27-Exosomes express membrane-bound p28, Ebi3 and CD81. (A) B-1a or CD19+ B-2 cells from mouse peritoneal cavity or spleen were stimulated with LPS or anti-IgM/anti-CD40 in exosomes-depleted medium for 72hrs (n=3-4/group). Exosomes in supernatant were purified and protein content in 5x106 exosomes was quantified by BCA protein assay (top panel) and amount of IL-27 secreted (pg/50µg exosome protein) was determined by ELISA (bottom panel) (n=3-4/group). (B-D) Exosomes isolated from supernatant of the BCR-activated B-1a cells were subjected to qPCR analysis (B) (n=6/group), Western blot analysis (C) (n=1/group) or reciprocal immunoprecipitation analysis (D). Controls in these analyses are exosome-depleted supernatant of B1a cell cultures. (E) Cell lysates and exosomes from activated B1a or B-2 cells were analyzed for IL-35 or IL-27 by ELISA(n=3/group). (F) For localization of IL-27 (p28 and Ebi3) and CD81 expression on the i27-Exosome membrane, the exosomes were captured with Tetraspanin Exo-Flow Capture Kit consisting of 9.1 µm diameter magnetic beads, stained with antibodies without fixation or permeabilization and then analyzed by confocal microscopy. The data are presented as the mean ± SD of at least three determinations, *p<0.05; **P < 0.01; ***P < 0.001; ****p<0.0001.

Article Snippet: Cells were then incubated with rabbit anti-CD81 (Cell Signaling), mouse anti-Ebi3 and rat anti-p28 (R&D system either MAB7430 or MAB18342).

Techniques: Membrane, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Isolation, Western Blot, Immunoprecipitation, Expressing, Magnetic Beads, Staining, Confocal Microscopy