Journal: EBioMedicine

Article Title: Quantification, epitope mapping and genotype cross-reactivity of hepatitis B preS-specific antibodies in subjects vaccinated with different dosage regimens of BM32

doi: 10.1016/j.ebiom.2020.102953

Figure Lengend Snippet: Comparison of preS-specific isotype and IgG subclasses responses in the four groups of patients. Shown are optical density (OD) values ( y -axes) corresponding to preS-specific IgG, IgA, IgM, IgE and IgG subclass (IgG 1 –IgG 4 ) levels in patients immunized with 3, 4 or 5 injections of BM32 or with placebo (see inlay) at baseline (V3), after one (V8) or four (V11) months treatment ( x -axes). Medians (horizontal bars) and significant differences are indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The detection was performed using either biotin-labeled mouse anti-human IgG 1 (BD, Cat. 555869) or biotin-labeled mouse anti-human IgG 4 (BD, Cat. 555882) in dilution 1:1000 mixed with HRP-conjugated streptavidin (BD, Cat. 554066) in dilution 1:2500 and incubated overnight at 4 °C.

Techniques:

Journal: EBioMedicine

Article Title: Quantification, epitope mapping and genotype cross-reactivity of hepatitis B preS-specific antibodies in subjects vaccinated with different dosage regimens of BM32

doi: 10.1016/j.ebiom.2020.102953

Figure Lengend Snippet: Quantification of IgG 1 and IgG 4 subclass responses in patients treated with placebo or different numbers of BM32 injections. Shown are absolute increases of serum concentrations of IgG 1 and IgG 4 ( y -axes: μg/ml) specific (a) for preS, (b) for peptide A, (c) for peptide B and (d) for peptide C from baseline (V3) to visits 8 and 11 ( x -axes) inpatients immunized with placebo, 3, 4 or 5 injections of BM32. Medians (horizontal bars) and significant differences are indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The detection was performed using either biotin-labeled mouse anti-human IgG 1 (BD, Cat. 555869) or biotin-labeled mouse anti-human IgG 4 (BD, Cat. 555882) in dilution 1:1000 mixed with HRP-conjugated streptavidin (BD, Cat. 554066) in dilution 1:2500 and incubated overnight at 4 °C.

Techniques:

Journal: EBioMedicine

Article Title: Quantification, epitope mapping and genotype cross-reactivity of hepatitis B preS-specific antibodies in subjects vaccinated with different dosage regimens of BM32

doi: 10.1016/j.ebiom.2020.102953

Figure Lengend Snippet: Kinetics and fine specificities of anti-preS antibody responses in the individual subjects of the placebo group, and the groups having received 3, 4 or 5 injections. The heat maps depict the log 10 of the serum concentrations of preS- or peptide-specific (a) IgG 1 and (b) IgG 4 . Missing values are indicated in gray.

Article Snippet: The detection was performed using either biotin-labeled mouse anti-human IgG 1 (BD, Cat. 555869) or biotin-labeled mouse anti-human IgG 4 (BD, Cat. 555882) in dilution 1:1000 mixed with HRP-conjugated streptavidin (BD, Cat. 554066) in dilution 1:2500 and incubated overnight at 4 °C.

Techniques:

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: Antibodies.

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: NP-LPS induces long-term and secondary antibody response in vivo. Three C57BL/6 mice (8-12 week old) were injected intraperitoneally with NP-LPS (25 μg) in PBS at d 0 and re-injected with NP-LPS (10 μg) in PBS at d 90 (second arrow). Sera were collected prior to the first injection and at different days, and analyzed for titers of NP3-binding IgG2b and IgG3 (depicted as relative units). Titers at d 0 were beyond the detection limit and set as 1 for the graph purpose.

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: In Vivo, Injection, Binding Assay

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: LPS induces B cells to undergo CSR and plasma cell differentiation in vitro in a dose- and time-dependent manner (flow cytometry analysis). (a, b) CSR to IgG1 in B cells stimulated with LPS at the indicated concentrations plus IL-4 for 4 d (a, top panels), and formation of B220loCD138hi plasma cells in the same cell culture (a, bottom panels). Mean and s.d. of data from four independent experiments were depicted in (b). p values calculated by paired student t test. (c) CSR to IgG1 in B cells stimulated with LPS plus IL-4, starting at d 0, and harvested at d 2, d 3, d 4, d 5 and d 7, as indicated. (d) Formation of B220loCD138hi plasma cells (top panels) as well as the proportion of IgG1+ (middle panels) and IgM+ (bottom panels) within those plasma cells in cultures of B cells stimulated with LPS plus IL-4, starting at d 0, and harvested at d 2, d 3, d 4, d 5 and d 7, as indicated. (e) CSR to IgG1 in B cells stimulated with LPS plus IL-4 for the period indicated, pelleted to remove LPS, and then cultured in RPMI-FBS only until being harvested at d 4. Data in (a, c, d, e) are representative of three independent experiments.

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: Cell Differentiation, In Vitro, Flow Cytometry, Cell Culture

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: LPS-induced CSR to IgG1 is enhanced by Pam3CSK4 and CpG in the presence of BCR signaling (flow cytometry analysis). (a) CSR to IgG1 in CFSE-labeled B cells stimulated with suboptimal doses of LPS, Pam3CSK4, R-848 and CpG (as indicated), alone or in pair-wise combination, all in the presence of anti–δ mAb/dex (100 ng/ml) and IL-4 for 4 d. (b) CSR to IgG1 in B cells stimulated with different concentrations of Pam3CSK4 and LPS in the presence of anti–δ mAb/dex (100 ng/ml) and IL-4 for 4 d.

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: Flow Cytometry, Labeling

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: LPS-induced CSR is inhibited by CpG and R-848 in the absence of BCR crosslinking (flow cytometry analysis). (a) The proportion of switched IgG1+IgM− B cells and IgM+IgG1− unswitched B cells in B cells after stimulation with LPS (3 μg/ml) plus IL-4 in the presence of nil, Pam3CSK4, R-848, CpG or GpC for 4 d. (b) CSR to IgG1 in CFSE-labeled B cells stimulated with LPS (3 μg/ml) plus IL-4 in the absence or presence of GpC (0.3 μM) or CpG (0.3 μM; left panels) for 4 d and depiction of the proportion of switched IgG1+ cells in B cells that had completed each cell division (right panels). (c) CSR to different IgG isotypes or IgA in B cells stimulated with LPS (3 μg/ml) plus appropriate cytokines (as indicated) in the absence or presence of CpG (0.3 μM) (mean and s.d. of data from three independent experiments; p values calculated by paired student t test). Data in (a) and (b) are representative of three independent experiments.

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: Flow Cytometry, Labeling

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: LPS-induced plasma cell differentiation and secretion of class-switched Ig are inhibited by CpG in the absence of BCR crosslinking. (a) Formation of B220loCD138hi plasma cells in cultures of B cells stimulated with LPS (1 μg/ml) plus IL-4 in the absence or presence of CpG (0.3 μM) for 4 d (flow cytometry analysis). (b) Formation of B220loCD138hi plasma cells in cultures of B cells stimulated with LPS plus IL-4, nil or IL-4, IL-5 and TGF-β, as indicated, in the absence or presence of CpG for 4 d (flow cytometry analysis). (c) ELISA of IgG1, IgG3 or IgA secreted into the supernatant of B cell cultures after stimulation with LPS plus IL-4, nil or IL-4, IL-5 and TGF-β, as indicated, in the absence or presence of CpG for 4 d. Data are mean and s.d. from three independent experiments (p values calculated by paired student t test).

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: Cell Differentiation, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: CD154-induced B cell class switching and differentiation into PNAhi B cells and plasma cells are inhibited by CpG in the absence of BCR crosslinking (flow cytometry analysis). (a) CSR to IgG1 (top panels) and expression of PNA-binding lectins (bottom panels) in B cells stimulated with CD154 (3 unit/ml) plus IL-4 in the absence or presence of CpG (0.3 μM) for 4 d. (b) CSR to IgG1 (top panels) and expression of PNA-binding lectins (bottom panels) in B cells stimulated with CD154 (3 unit/ml) plus IFN-γ in the absence or presence of CpG for 4 d. (c) CSR to IgG1, IgE or IgG2a in B cells stimulated with CD154 plus IL-4 or IFN-γ in the absence or presence of CpG for 4 d. Data are mean and s.d. from three independent experiments (p values calculated by paired student t test). (d) Formation of B220loCD138hi plasma cells in cultures of B cells stimulated with LPS (1 μg/ml) plus IL-4 or IFN-γ in the absence or presence of CpG (0.3 μM) for 4 d. Data are mean and s.d. from three independent experiments (p values calculated by paired student t test).

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: Flow Cytometry, Expressing, Binding Assay

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: CpG-mediated inhibition of LPS- or CD154-induced CSR requires co-stimulation of B cells by CpG with LPS or CD154. (a) Levels of transcripts, as indicated, in B cells stimulated with CpG plus IL-4 for 0, 4 or 24 h (real-time qRT-PCR analysis). (b, c) CSR to IgG1 in CFSE-labeled B cells stimulated with LPS (3 μg/ml, b) or CD154 (3 unit/ml, c) plus CpG (0.3 mM) for the period as indicated plus IL4 (flow cytometry analysis). Data are representative of three independent experiments.

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: Inhibition, Quantitative RT-PCR, Labeling, Flow Cytometry

Journal: Autoimmunity

Article Title: B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

doi: 10.3109/08916934.2014.993027

Figure Lengend Snippet: Co-stimulation of B cells by LPS and CD154 hampers CSR and plasma cell differentiation (flow cytometry analysis). (a, b) CSR to IgG1 (a) and formation of B220loCD138hi plasma cells (b) in B cell cultures after stimulation with LPS (3 μg/ml), CD154 (3 unit/ml) or both plus IL-4 for 4 d. Data are representative of three independent experiments.

Article Snippet: Applications Mouse B220, PE-conjugated Rat mAb eBioscience 25-0452-83 FCM Mouse B220, APC-conjugated Rat mAb BD Pharmingen 553092 FCM Mouse CD138, biotin-conjugated Rat mAb BD Pharmingen 553713 FCM Mouse Ig Goat pAb Southern Biotech 1012-01 ELISA Mouse IgG Goat pAb Southern Biotech 1030-01 ELISA Mouse IgM, FITC-conjugated Rat mAb BD Pharmingen 553437 FCM Mouse IgG1, FITC-conjugated Rat mAb BD Pharmingen 553443 FCM Mouse IgG1, APC-conjugated Rat mAb BD Pharmingen 550874 FCM Mouse IgG1, biotin-conjugated Goat pAb Southern Biotech 1070-08 ELISA Mouse IgG2a, FITC-conjugated Rat mAb BD Pharmingen 553390 FCM Mouse IgG2b, biotin-conjugated Goat pAb Southern Biotech 1090-08 FCM Mouse IgG3, FITC-conjugated Rat mAb BD Biosciences 553403 FCM Mouse IgG3, biotin-conjugated Goat pAb Southern Biotech 1100-08 ELISA Mouse IgA, FITC-conjugated Rat mAb BD Pharmingen 559354 FCM Mouse IgA, biotin-conjugated Goat pAb Southern Biotech 1040-08 FCM, ELISA Mouse IgE, PE-conjugated Rat mAb eBioscience Dec-92 FCM Open in a separate window Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody; FCM, flow cytometry; ELISA, enzyme-linked immunosorbent assay.

Techniques: Cell Differentiation, Flow Cytometry

Journal: Stem Cells and Development

Article Title: Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways

doi: 10.1089/scd.2016.0199

Figure Lengend Snippet: Preparation of monocyte-derived dendritic cells (DC). (A) Plastic-adherent PBMC (>80% CD14+) was differentiated in DC medium for 5–7 days. (B) Greater than 95% of high forward versus side scatter cells in the DC differentiation culture expressed high levels of CD11c and MHCII. DC derived from adherent monocytes (see Materials and Methods section) were defined as large cells (relative high forward and side scatter profiles by flow cytometry) expressing high levels of surface CD11c and MHCII. (C) Cells expressing high surface CD11c readily phagocytosed soluble ovalbumin (OVA) antigen. Fluorescence microscopy showing cells with high surface expression of CD11c (arrow), and “OVA” arrow indicates APC-conjugated OVA that has been endocytosed by CD11chi cells. Similar results were obtained in >10 additional experiments. PBMC, peripheral blood mononuclear cell.

Article Snippet: Adherent cells were between 70% and 85% CD14 + identified by flow cytometry using the Alexa Fluor 647-conjugated (Tük4 clone, IgG2a) canine cross-reactive CD14 antibody ( ) ( www.bio-rad-antibodies.com/human-cd14-antibody-tuk4-mca1568.html ).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Fluorescence, Microscopy

Journal: Nature immunology

Article Title: The FcμR Limits Tonic BCR Signaling by Regulating IgM-BCR Expression

doi: 10.1038/ni.3677

Figure Lengend Snippet: Concentrations of ( a ) IgM, ( b ) IgA, ( c ) IgG in Fcmr flx/flx Cd19 -Cre and control sera from 3 months old mice (n=10–18 mice/group). ( d ) Frequencies of IgM and ( e ) IgG secreting cells in spleens and BMs of these mice (n=4 mice/group). ( f ) Relative amounts of IgM and ( g ) IgG anti-dsDNA and relative amounts of anti-dsDNA/mg Ig per μg total Ig in Fcmr flx/flx Cd19 -Cre and control sera (n=10–12 mice/group). ( h ) Overlay histograms of Fas expression by B cells from indicated mice (left). Graph summarizing results (right) (n=2 mice/group). ( i ) 5% contour plots with outliners gated on live CD19 + B cells. Gate and numbers identify CD38 low PNA + germinal center (GC) B cells. Graphs summarize frequencies ± SD GC B cells of total cells in Fcmr flx/flx Cd19 -Cre and control spleens (n=4–5 mice/group). ( j ) Relative amounts influenza A/Puerto Rico/8/34-binding IgM in sera from naïve Fcmr flx/flx Cd19 -Cre and control mice (n=15–18 mice/group). ( k ) Relative amounts of influenza A/PR8-binding and ( l ) total IgM in sera of Fcmr flx/flx Cd19 -Cre, and control mice one day after A/PR8 infection (n=8, 14 mice/group). Data are representative of at least two independent experiments. ( m ) Lung viral loads at 1 day after A/PR8 infection. For Figure k , l and m data are combined from three independent experiments (mean ± SD in a–m) . *p<0.05, **p<0.005, ***p<0.0005 by unpaired two-tailed Student’s t -test.

Article Snippet: For PI3K-p85 subunit staining, fixed and permeabilized cells were stained with anti-p85 (Abcam ab189403, 6G10), anti mouse IgG1-biotin (SouthernBiotech,1070-05), and with SA-Qdot 605 for 30 min at each step.

Techniques: Control, Expressing, Binding Assay, Infection, Two Tailed Test

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: (A, B) Determination of IC 50 values in a competition ELISA for compound binding to human and cyno IL-7Rα and γc. The assay format is IL-7Rα-(His) 6 -tagged ECD or Fc-γc ECD immobilized on 96-well plates. Tracers are C-terminal biotinylated forms of the reference IL-7Rα and γc peptide ligands, each pre-complexed with Neutravidin-HRP (NA-HRP). (C, D) Label-free measurement of the equilibrium dissociation constant (K D ) for peptide binding to IL-7Rα and γc extracellular domains with peptides was performed by biolayer interferometry as detailed in methods. Red lines depict 1:1 binding fit from Gator™ software. Blue lines represent individual BLI sensogram data taken every 0.1 seconds. Inset table displays comparison of ELISA data and BLI kinetic data.

Article Snippet: Biotinylated mouse anti-human IgG2 Fc (Southern Biotech Cat# 9070–08) was used as the capture antibody, and the rabbit polyclonal antibody against MDK1188, followed by a mouse anti-rabbit IgG Fc HRP (Genscript Cat# A01856-200) as the detection antibody.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Software, Comparison