Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppressive effect of bacterial polysaccharides on BAFF system is responsible for their poor immunogenicity.

doi: 10.4049/jimmunol.1002976

Figure Lengend Snippet: FIGURE 1. Ig secretion and plasma cell gen- eration from MCPS-treated B cells. A, MCPS or NP-Ficoll concentrations, ranging from 2.5 to 40 mg/ml, were used to prestimulate B cells followed by a restimulation with BAFF and IL- 4 or APRIL and IL-4 after 24 h. LPSs with IL-4 or TGF-b were included as positive controls for IgG and IgA switch, respectively. Culture supernatants were harvested after 6 d, and Ig levels were measured by ELISA. *p , 0.05, in the difference between media-pretreated and MCPS- or NP-Ficoll–pretreated B cell culture supernatant Ig levels. Data shown represent the average of three experiments. B, MCPS, rang- ing from 2.5 to 40 mg/ml, was used to co- stimulate B cells with BAFF and anti-IgM or APRIL and anti-IgM for 72 h, after which cells were stained for CD138 and B220. Experiment shown is representative of three experiments with similar results.

Article Snippet: Abs against mouse cell markers and the isotype controls used in flow cytometry assay were TACI-PE, BCMA-FITC, BAFF-PE, Rat IgG2a-PE, Rat IgG1-FITC (R&D Systems), CD21-FITC, CD21-PE, CD23-APC-Cy7, CD23-Biotin, CD5APC, IgM-APC, B220/CD45R-PE-Cy5, B220/CD45R-Pacific Blue, CD3FITC, CD138-APC, CD19-FITC, CD40-APC, MHC-I-PE, MHC-II-FITC, CD80-FITC and CD86-PE, Rat IgG2b-FITC, Rat IgG2b-PE, Rat IgG2aBiotin, Rat IgG2a-APC, Rat IgG2a-PE-Cy5, Rat IgG2a-Pacific Blue, Streptavidin-APC-CY7, CD11c-APC, Armenian hamster IgG1 l2-APC, purified rat anti-mouse CD16/CD32 (BD Biosciences, Pharmingen, San Jose, CA), BAFFR-FITC, IgM-PECy7, Rat IgG1-FITC, Rat IgG2a-PECy7 (eBioscience), B220/CD45R Alexa Fluor 405, and IgG2a-Alexa Fluor 405 (Invitrogen).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppressive effect of bacterial polysaccharides on BAFF system is responsible for their poor immunogenicity.

doi: 10.4049/jimmunol.1002976

Figure Lengend Snippet: FIGURE 9. The expression of TACI and BAFFR and secretion of Ig from B cells costimulated with CpG and MCPS. As indicated, MCPS ranging from 2.5 to 40 mg/ml was used to stimulate B cells along with CpG for 24 h, and receptor expression was determined after staining for TACI/B220 or BAFFR/ B220 by flow cytometry (A). Equal numbers of cells from each condition were pooled together to stain for isotype control. Data shown represent one experi- ment of three experiments. B, Purified B cells, pres- timulated with 40 mg/ml of MCPS and CpG ODN for 24 h, were restimulated with BAFF and IL-4 or APRIL and IL-4; LPSs with IL-4 or TGF-b were included as positive controls for the IgG and IgA switch, respectively. Culture supernatants were har- vested after 6 d, and Ig levels were measured by ELISA. Data shown represent an average of three experiments. *p , 0.05, **p , 0.01, ***p , 0.0001.

Article Snippet: Abs against mouse cell markers and the isotype controls used in flow cytometry assay were TACI-PE, BCMA-FITC, BAFF-PE, Rat IgG2a-PE, Rat IgG1-FITC (R&D Systems), CD21-FITC, CD21-PE, CD23-APC-Cy7, CD23-Biotin, CD5APC, IgM-APC, B220/CD45R-PE-Cy5, B220/CD45R-Pacific Blue, CD3FITC, CD138-APC, CD19-FITC, CD40-APC, MHC-I-PE, MHC-II-FITC, CD80-FITC and CD86-PE, Rat IgG2b-FITC, Rat IgG2b-PE, Rat IgG2aBiotin, Rat IgG2a-APC, Rat IgG2a-PE-Cy5, Rat IgG2a-Pacific Blue, Streptavidin-APC-CY7, CD11c-APC, Armenian hamster IgG1 l2-APC, purified rat anti-mouse CD16/CD32 (BD Biosciences, Pharmingen, San Jose, CA), BAFFR-FITC, IgM-PECy7, Rat IgG1-FITC, Rat IgG2a-PECy7 (eBioscience), B220/CD45R Alexa Fluor 405, and IgG2a-Alexa Fluor 405 (Invitrogen).

Techniques: Expressing, Staining, Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppressive effect of bacterial polysaccharides on BAFF system is responsible for their poor immunogenicity.

doi: 10.4049/jimmunol.1002976

Figure Lengend Snippet: FIGURE 11. The effect of GBS-V on the BAFF system. A, Purified mouse splenic B cells were in- cubated with 40 mg/ml MCPS or GBS-V for 24 h and stained with Abs against TACI and BAFFR along with B220. Data shown represent one of three experiments with similar results. B, MCPS or GBS-V, each at 40 mg/ ml concentration, was used to prestimulate B cells. At 24 h, cells were restimulated with BAFF and IL-4 or APRIL and IL-4; LPSs with IL-4 or TGF-b were in- cluded as positive controls for the IgG and IgA switch respectively. Culture supernatants were harvested after 6 d, and Ig levels were measured by ELISA. Data shown represent an average of three experiments. The p values are derived from the comparison of poly- saccharide-pretreated cells with media-pretreated cells. *p , 0.001, **p , 0.01, ***p , 0.05. C, MCPS or GBS-V (both at 40 mg/ml concentration) was used to stimulate splenic B cells for 24 h, and cell death was determined by Annexin V/7-AAD staining in flow cytometry. Data from a representative experiment of three experiments with similar results are shown. D, Percent live (Annexin Vneg and 7-AADneg) and dead cells (Annexin Vpos and 7-AADpos) following MCPS and GBS-V incubation. Average percentages from three experiments are plotted. *p , 0.05 compared with media-treated cells; **p , 0.05, media-treated live cells. E, Concentration of BAFF in the culture supernatants of bone marrow-derived DCs stimulated for 24 h with indicated CPS, with or without IFN-g. *p , 0.05.

Article Snippet: Abs against mouse cell markers and the isotype controls used in flow cytometry assay were TACI-PE, BCMA-FITC, BAFF-PE, Rat IgG2a-PE, Rat IgG1-FITC (R&D Systems), CD21-FITC, CD21-PE, CD23-APC-Cy7, CD23-Biotin, CD5APC, IgM-APC, B220/CD45R-PE-Cy5, B220/CD45R-Pacific Blue, CD3FITC, CD138-APC, CD19-FITC, CD40-APC, MHC-I-PE, MHC-II-FITC, CD80-FITC and CD86-PE, Rat IgG2b-FITC, Rat IgG2b-PE, Rat IgG2aBiotin, Rat IgG2a-APC, Rat IgG2a-PE-Cy5, Rat IgG2a-Pacific Blue, Streptavidin-APC-CY7, CD11c-APC, Armenian hamster IgG1 l2-APC, purified rat anti-mouse CD16/CD32 (BD Biosciences, Pharmingen, San Jose, CA), BAFFR-FITC, IgM-PECy7, Rat IgG1-FITC, Rat IgG2a-PECy7 (eBioscience), B220/CD45R Alexa Fluor 405, and IgG2a-Alexa Fluor 405 (Invitrogen).

Techniques: Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Comparison, Cytometry, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppressive effect of bacterial polysaccharides on BAFF system is responsible for their poor immunogenicity.

doi: 10.4049/jimmunol.1002976

Figure Lengend Snippet: FIGURE 10. Ag-specific IgG response in MCPS- and NP-Ficoll– injected BALB/c mice. MCPS (dose) or NP-Ficoll (dose) with or without CpG ODN (dose) was used to vaccinate mice. PBS-injected mice were included as controls. Day 15 serum anti–NP-Ficoll and anti-MCPS IgG Ab levels are plotted. Other than the PBS-injected mice group, which con- tained four mice, each vaccine group contained five mice. Data shown represent average OD values from one experiment. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Abs against mouse cell markers and the isotype controls used in flow cytometry assay were TACI-PE, BCMA-FITC, BAFF-PE, Rat IgG2a-PE, Rat IgG1-FITC (R&D Systems), CD21-FITC, CD21-PE, CD23-APC-Cy7, CD23-Biotin, CD5APC, IgM-APC, B220/CD45R-PE-Cy5, B220/CD45R-Pacific Blue, CD3FITC, CD138-APC, CD19-FITC, CD40-APC, MHC-I-PE, MHC-II-FITC, CD80-FITC and CD86-PE, Rat IgG2b-FITC, Rat IgG2b-PE, Rat IgG2aBiotin, Rat IgG2a-APC, Rat IgG2a-PE-Cy5, Rat IgG2a-Pacific Blue, Streptavidin-APC-CY7, CD11c-APC, Armenian hamster IgG1 l2-APC, purified rat anti-mouse CD16/CD32 (BD Biosciences, Pharmingen, San Jose, CA), BAFFR-FITC, IgM-PECy7, Rat IgG1-FITC, Rat IgG2a-PECy7 (eBioscience), B220/CD45R Alexa Fluor 405, and IgG2a-Alexa Fluor 405 (Invitrogen).

Techniques: Injection