il 12p35 pe  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Faecalibacterium prausnitzii Skews Human DC to Prime IL10-Producing T Cells Through TLR2/6/JNK Signaling and IL-10, IL-27, CD39, and IDO-1 Induction

doi: 10.3389/fimmu.2019.00143

Figure Lengend Snippet: DC exposure to F. prausnitzii switches their cytokine profile from pro-inflammatory to anti-inflammatory. IL-12p70 (A) , TNF-a (B) , and IL-10 levels (C) secreted in response to LPS by DC exposed or not (Ctrl) to Fprau at the beginning of their differentiation. (D) Representative dot plot of intracellular IL-12p35 and p40 co-labeling in LPS-stimulated DC exposed or not to F. prausnitzii . (E) LPS-induced IL-12p40 ( n = 6–24) and IL-12p35 ( n = 9–27) expression by DC exposed or not (Ctrl) to indicated bacteria (ratio:1:1) during the last 48 h. (F) IL-12p40 ( n = 12) and p35 ( n = 6) expression by myeloid DC maintained for 24 h in culture with or without F. prausnitzii , and then stimulated 12 h with LPS. Wilcoxon test. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005.

Article Snippet: We used: PE-labeled antibodies to CD40 (clone 5C3), CD80 (clone L307.4), CD83 (clone HB15e), CD39 (clone TU66), PDL-1 (clone MIH1), as well as anti-CD4-APC (clone SK3) all from Becton Dickinson: (BD), anti-CD86-PE (clone HA5.2B7, Beckman Coulter), anti-IL-12p35/p70-PE (clone REA121, Miltenyi Biotech), anti-IL-12/IL-23p40-APC (clone C11.5, BioLegend), anti-IL-10-PE (clone JES3-19F1, BD), anti-IFN-γ-APC (clone B27, BD), anti-IL-13-PE (clone JES10-5A2, BD), anti-IDO-1-APC (clone 70083, R&D), anti-HO-1 (HO-1-1, Thermoscientific).

Techniques: Labeling, Expressing, Bacteria

Journal: The Journal of Clinical Investigation

Article Title: ST8Sia6 overexpression protects pancreatic β cells from spontaneous autoimmune diabetes in nonobese diabetic mice

doi: 10.1172/JCI181207

Figure Lengend Snippet: ( A and B ) Normalized cell count of classical macrophages (CD11b + CD11c – F4/80 + Gr1 – ), hybrid macrophages (CD11b + CD11c + F4/80 + Gr1 – ), cDC1s (CD11b – CD11c + F4/80 – Gr1 – ), and cDC2s (CD11b + CD11c + F4/80 – Gr1 – ) per islet ( A ) or per pancreatic lymph node ( B ) from euglycemic 14-week-old NOD βST or littermate mice. ( C ) Representative flow cytometry histogram of IL-12p35 expression in cDC2s from islets of euglycemic 14-week-old NOD βST and NOD littermate mice, compared with isotype control. ( D ) Representative flow cytometry histogram of IL-12p35 expression in hybrid macrophages from islets of euglycemic NOD βST and littermate mice, compared with isotype control. Vertical line denotes the positive gate. ( E ) Quantification of IL-12p35 expression shown in C and D across 7 NOD βST mice, 16 NOD littermate mice, and 5 isotype controls. Error bars represent SD. Mann-Whitney U tests were used to examine statistical differences between groups.

Article Snippet: The following antibodies were used: CD45-BUV395 (BD 564279 at 1:1,000), L/D-UV450 (Tonbo 13-0868 at 1:1,000), CD11c-BUV615 (BD 751222 at 1:100), CD11b-PacBlue (BioLegend 562894 at 1:200), Ly6C-BV480 (BioLegend 569439 at 1:500), CD8b-BV785 (BioLegend 126631 at 1:1,000), Ly6G-BV785 (BioLegend 127645 at 1:200), CD4-SparkBlue (BioLegend 100494 at 1:1,000), T-bet–RB780 (BD 569089 at 1:100), F4/80-ef660 (Invitrogen 50-4801-82 at 1:200), IFN-γ–APC (BioLegend 505810 at 1:100), isotype control–APC (BD 554686 at 1:100), FoxP3-APC (Tonbo 20-5773 at 1:100), CD11c-BV421 (BioLegend 117330 at 1:500), RORγt-BV421 (BD 562894 at 1:100), MHCII-BV510 (BioLegend 107635 at 1:400), L/D-BV510 (Tonbo 13-0870 at 1:1,000), CD8a-BV510 (BioLegend 100752 at 1:500), TCRβ-BV605 (BioLegend 109241 at 1:200), CD45-BV785 (BioLegend 103149 at 1:500), CD8a-BV785 (BioLegend 100750 at 1:500), CD11b-FITC (BioLegend 101206 at 1:200), CD45-FITC (BioLegend 103108 at 1:500), L/D-GR780 (Tonbo 13-0865 at 1:1,000), IL-12p35–PE (R&D Systems IC2191P at 1:100), isotype control–PE (BioLegend 400408 at 1:100), Siglec-E–PE–Cy7 (BioLegend 677108 at 1:100), isotype control–PE–Cy7 (BioLegend 400522 at 1:100), T-bet–PE–Cy7 (BioLegend 644824 at 1:500), F4/80-PE-Dazzle (BioLegend 123146 at 1:400), Gr1-PerCP (BioLegend 108426 at 1:500), CD4-PerCP (BioLegend 100538 at 1:500), PD-1–APC–Fire750 (BioLegend 135240 at 1:200), and PD-L1–BV421 (BioLegend 124315 at 1:100).

Techniques: Cell Counting, Flow Cytometry, Expressing, Control, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: ST8Sia6 overexpression protects pancreatic β cells from spontaneous autoimmune diabetes in nonobese diabetic mice

doi: 10.1172/JCI181207

Figure Lengend Snippet: ( A ) Confirmation of ST8Sia6-myc shutoff after 5 weeks of doxycycline treatment by IHC. Schematic of doxycycline treatment for temporal control of ST8Sia6 expression. Scale bars: 100 μm. ( B ) Diabetes-free incidence after treatment of 15 euglycemic NOD βST and 29 euglycemic littermate mice with doxycycline starting at 8 weeks of age. ( C ) Comparison with disease kinetics of 8-week-old euglycemic non-doxycycline-treated NOD βST ( n = 50) or littermate ( n = 151) mice (subset from ). ( D ) Diabetes-free incidence after treatment of 12 euglycemic NOD βST and 16 euglycemic littermate mice with doxycycline starting at 20 weeks of age. ( E ) Comparison with disease kinetics of 20-week-old euglycemic non-doxycycline-treated NOD βST ( n = 50) or littermate ( n = 115) mice (subset from ). Log-rank Mantel-Cox test was used to analyze statistical differences in diabetes-free incidence of indicated groups. ( F ) Insulitis distribution in pancreas sections of NOD βST mice treated with doxycycline for 5 weeks from 8 weeks or 20 weeks of age, compared with 300-day-old pancreata from NOD βST mice never treated with doxycycline (subset from ). Scoring and analysis as in . n = 69 islets from 12 mice (8-week doxycycline) or 127 islets from 11 mice (20-week doxycycline). ( G and H ) Ratio of T-bet + Tregs (T-bet + FoxP3 + CD4 + ) to Th1 T cells (T-bet + FoxP3 – CD4 + ) ( G ) or to SLECs (T-bet + CD8 + ) ( H ) from islets of 20-week-old mice treated with doxycycline for greater than 5 weeks. ( I ) Quantification of IL-12p35 expression in hybrid macrophages (CD11b + CD11c + F4/80 + Gr1 – ) and cDC2s (CD11b + CD11c + F4/80 – Gr1 – ) from islets of 20-week-old mice treated with doxycycline for greater than 5 weeks. Same gating schemes as in and 5. Error bars represent SD. Mann-Whitney U test was used for statistical analysis between groups.

Article Snippet: The following antibodies were used: CD45-BUV395 (BD 564279 at 1:1,000), L/D-UV450 (Tonbo 13-0868 at 1:1,000), CD11c-BUV615 (BD 751222 at 1:100), CD11b-PacBlue (BioLegend 562894 at 1:200), Ly6C-BV480 (BioLegend 569439 at 1:500), CD8b-BV785 (BioLegend 126631 at 1:1,000), Ly6G-BV785 (BioLegend 127645 at 1:200), CD4-SparkBlue (BioLegend 100494 at 1:1,000), T-bet–RB780 (BD 569089 at 1:100), F4/80-ef660 (Invitrogen 50-4801-82 at 1:200), IFN-γ–APC (BioLegend 505810 at 1:100), isotype control–APC (BD 554686 at 1:100), FoxP3-APC (Tonbo 20-5773 at 1:100), CD11c-BV421 (BioLegend 117330 at 1:500), RORγt-BV421 (BD 562894 at 1:100), MHCII-BV510 (BioLegend 107635 at 1:400), L/D-BV510 (Tonbo 13-0870 at 1:1,000), CD8a-BV510 (BioLegend 100752 at 1:500), TCRβ-BV605 (BioLegend 109241 at 1:200), CD45-BV785 (BioLegend 103149 at 1:500), CD8a-BV785 (BioLegend 100750 at 1:500), CD11b-FITC (BioLegend 101206 at 1:200), CD45-FITC (BioLegend 103108 at 1:500), L/D-GR780 (Tonbo 13-0865 at 1:1,000), IL-12p35–PE (R&D Systems IC2191P at 1:100), isotype control–PE (BioLegend 400408 at 1:100), Siglec-E–PE–Cy7 (BioLegend 677108 at 1:100), isotype control–PE–Cy7 (BioLegend 400522 at 1:100), T-bet–PE–Cy7 (BioLegend 644824 at 1:500), F4/80-PE-Dazzle (BioLegend 123146 at 1:400), Gr1-PerCP (BioLegend 108426 at 1:500), CD4-PerCP (BioLegend 100538 at 1:500), PD-1–APC–Fire750 (BioLegend 135240 at 1:200), and PD-L1–BV421 (BioLegend 124315 at 1:100).

Techniques: Control, Expressing, Comparison, MANN-WHITNEY

Journal: Nutrients

Article Title: Tualang Honey Has a Protective Effect Against Photodamage and Skin Cancer: An In Vivo Study

doi: 10.3390/nu16244314

Figure Lengend Snippet: TH 0.1% increases IL-12p35- and IL-23p19-producing CD11c cells in UVB-irradiated mice. Percentages of IL-12p35- and IL-23p19-producing CD11c+ cells in draining lymph nodes were analyzed by flow cytometry 24 h post-UVB exposure. Data represent mean ± SD for five animals per group (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant). TH, tualang honey; UVB, ultraviolet B.

Article Snippet: Monoclonal antibodies used for flow cytometry studies included anti-mouse CD11b (M1-70 PerCp), Gr-1 (RB6-8C5; AF488), CD11c (N418; APC), MHC II (M5 114.15.2; AF700), IL-12p35 (27537; PE), and IL-23p19 (fc23cpg; AF488) (Thermofisher Scientific, Waltham MA, USA).

Techniques: Irradiation, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: RANK Ligand Helps Immunity to Leishmania major by Skewing M2-Like Into M1 Macrophages

doi: 10.3389/fimmu.2020.00886

Figure Lengend Snippet: RANKL induces a M2-like to M1 phenotype shift. Inflammatory (B6) macrophages were cultured in triplicates with medium or RANKL in the presence or absence of IFN-γ. After 48 h, cells were harvested and stained with anti-F4/80, anti-CD301 (MGL), anti-IL-12p35, or control mAbs. (A) Plots depict F4/80 + cells as evaluated for CD301 (MGL) and intracellular IL-12p35 expression. (B) Results are expressed as means and S.E.M. Significant differences between macrophages treated with or without RANKL were analyzed by t -test and indicated for * P <0.05, and ** P <0.01. Data are representative of 2 independent experiments.

Article Snippet: For intracellular staining, we washed, permeabilized, fixed, and stained cells with PE-labeled anti-IL-12p35 or control murine IgG1 mAb (R&D Systems), PE-labeled anti-NOS2 (iNOS) or control rat IgG2a mAb (eBioscience); FITC-labeled anti-Arginase-1 or control sheep IgG mAb (R&D Systems).

Techniques: Cell Culture, Staining, Expressing