Spleen and PeC CD19+ B cells (3 × 106 cells/well) stimulated for 5 or 48 h with LPS (10 μg/ml, Sigma-Aldrich, St. Louis, MO). The B cells were incubated with LPS alone or with JQ1 (0, 20, 50, and 100 nM) for 5 h or indicated times in figure legends, and phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, Sigma-Aldrich), ionomycin (500 ng/ml, Sigma-Aldrich), and Monensin (2 μM, eBioscience, San Diego, CA) were added during last 5 h. Prior to surface staining, Fcγ receptors were blocked with anti-CD16/CD32 monoclonal antibodies (2.4G2, BD Biosciences). Cells were fixed and permeabilized with a Cytofix/Cytoperm kit (eBioscience) and then were stained with anti-IL-10 (JES5-16E3, eBioscience) (46 (link)). The antibodies against surface proteins were as follows: CD1d (1B1), CD5 (53-7.3), CD11b (M1/70), CD19 (eBio1D3), CD21/CD35 (eBioBD9), CD23 (B3B4), CD40 (HM40-3), CD86 (GL1), B220 (RA3-6B2), IgM (eB121-15F9), IgD (11 (link)–26 (link)), MHCII (M5/114.15.2), all purchased from eBioscience. Cells were analyzed with FACSCanto II (BD Biosciences, San Jose, CA) and FlowJo V10 software (TreeStar, Ashland, OR). The gate strategy for IL-10-producing B cells was illustrated in Supplementary Fig. 3 .
Protocol detail
B cell Stimulation and IL-10 Analysis
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Other organizations : Konkuk University, Konkuk University Medical Center, Duksung Women's University
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Variable analysis
independent variables
- LPS concentration (0, 10 μg/ml)
- JQ1 concentration (0, 20, 50, 100 nM)
- Stimulation duration (5 h, 48 h)
- IL-10 production by CD19+ B cells
- Cell type (Spleen and PeC CD19+ B cells)
- Cell number (3 × 10^6 cells/well)
- PMA, ionomycin, and Monensin treatment (50 ng/ml, 500 ng/ml, and 2 μM, respectively, added during the last 5 h)
- Fcγ receptor blocking with anti-CD16/CD32 antibodies
- Fixation and permeabilization using Cytofix/Cytoperm kit
- Antibodies for surface staining (CD1d, CD5, CD11b, CD19, CD21/CD35, CD23, CD40, CD86, B220, IgM, IgD, MHCII)
- Flow cytometry analysis (FACSCanto II and FlowJo V10 software)
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