Journal: PLoS ONE

Article Title: Early decreased TLR2 expression on monocytes is associated with their reduced phagocytic activity and impaired maturation in a porcine polytrauma model

doi: 10.1371/journal.pone.0187404

Figure Lengend Snippet: ( A ). Cells were characterized by CD14 and TLR2 expression by flow cytometry. Blood samples were collected from polytraumatized pigs (n = 12) before trauma as baseline, immediately after surgery/trauma, 3.5 h, 5.5 h, 24 h and 72 h after trauma. The percentage of phagocytizing porcine peripheral monocytes out of total monocytes was determined ( B ). Whole blood samples were incubated with pHrodo® Red S . aureus BioParticles® Conjugate. The measurement was performed by flow cytometry. Monocytes were discriminated by forward and sideward scatter. Data are shown as mean ± sem. *: p <0.05 vs . baseline (before surgery). Data are shown as mean ± sem. *: p <0.05 vs . baseline (before surgery).

Article Snippet: The samples were kept at room temperature and incubated for one hour at room temperature in darkness with the rat IgG polyclonal neutralizing human TLR2 (Invivogen) (20 μg/ml).

Techniques: Expressing, Flow Cytometry, Incubation

Journal: PLoS ONE

Article Title: Early decreased TLR2 expression on monocytes is associated with their reduced phagocytic activity and impaired maturation in a porcine polytrauma model

doi: 10.1371/journal.pone.0187404

Figure Lengend Snippet: Blood samples were collected from healthy pigs (n = 10). Whole blood samples were incubated in the first step with TLR2 neutralizing (nAB TLR2) or control (nAB ctrl) antibody, or without antibody (ctrl). In the next step, the samples were incubated with pHrodo® Red S . aureus BioParticles® Conjugate. The measurement was performed by flow cytometry. Monocytes were discriminated by forward and sideward scatter. Data are shown as mean ± sem. *: p <0.05 vs . ctrl.

Article Snippet: The samples were kept at room temperature and incubated for one hour at room temperature in darkness with the rat IgG polyclonal neutralizing human TLR2 (Invivogen) (20 μg/ml).

Techniques: Incubation, Flow Cytometry

Journal: The Journal of allergy and clinical immunology

Article Title: TLR2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation

doi: 10.1016/j.jaci.2016.01.037

Figure Lengend Snippet: Primer sequences for real-time quantitative PCR.

Article Snippet: Selected cultures were also incubated with 50 ng/ml IL-4 (Peprotech), 30 μg/ml monoclonal anti-TLR2 neutralizing antibody (clone T2.5, eBioscience, San Diego, CA) or isotype control antibody Animals C57BL/6 mice WT and TLR2−/− (B6.129-Tlr2 tm1Kir /J TLR2 KO) were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques:

Journal: The Journal of allergy and clinical immunology

Article Title: TLR2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation

doi: 10.1016/j.jaci.2016.01.037

Figure Lengend Snippet: A. RV1B infection of bone marrow-derived macrophages from wild type C57BL/6 and TLR2−/− mice. B, C. RV infection of human macrophages derived from CD14+ peripheral blood monocytes. Macrophages were cultured the presence or absence of IL-4 and infected with sucrose gradient-purified RV1B (B) or RV39 (C). Whole lung mRNA expression was measured by quantitative PCR. Signals were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative CT (2−ΔΔCT) method and expressed as fold-change versus GAPDH. (N=3 samples per condition, mean±SEM, *different from wild type or IgG control, p<0.05, ANOVA.)

Article Snippet: Selected cultures were also incubated with 50 ng/ml IL-4 (Peprotech), 30 μg/ml monoclonal anti-TLR2 neutralizing antibody (clone T2.5, eBioscience, San Diego, CA) or isotype control antibody Animals C57BL/6 mice WT and TLR2−/− (B6.129-Tlr2 tm1Kir /J TLR2 KO) were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Infection, Derivative Assay, Cell Culture, Purification, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of allergy and clinical immunology

Article Title: TLR2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation

doi: 10.1016/j.jaci.2016.01.037

Figure Lengend Snippet: Wild-type and TLR2−/− mice infected with intranasally with RV or sham HeLa cell lysate control, sacrificed and the lungs harvested at different time points. A. RV positive strand RNA was assessed 24–72 h after infection and normalized by total μg of RNA. B. Transcript levels of TLR2 from lungs of mice infected with RV. C. BAL neutrophils. D–G. Transcript levels of pro-inflammatory cytokines CXCL1 and TNF-α (D, E) and the type I interferons IFN-α and IFN-β (F, G) were assessed by quantitative PCR and results expressed as fold change over GADPH. (For A–G, N=3 mice per group, mean±SEM, *different from wild type, p<0.05, ANOVA.) H. Hematoxylin and eosin staining of lung sections from sham and RV-infected mice harvested 24 h post-inoculation. Magnification, 200X.

Article Snippet: Selected cultures were also incubated with 50 ng/ml IL-4 (Peprotech), 30 μg/ml monoclonal anti-TLR2 neutralizing antibody (clone T2.5, eBioscience, San Diego, CA) or isotype control antibody Animals C57BL/6 mice WT and TLR2−/− (B6.129-Tlr2 tm1Kir /J TLR2 KO) were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Infection, Real-time Polymerase Chain Reaction, Staining

Journal: The Journal of allergy and clinical immunology

Article Title: TLR2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation

doi: 10.1016/j.jaci.2016.01.037

Figure Lengend Snippet: A–D. Female C57BL/6 wild-type or TLR2 null mice were exposed to either PBS or OVA before infection with RV or sham control for 24 h. BAL neutrophils (A) and eosinophils (B). (N=3 mice per group, mean±SEM, *different from wild type, †different from OVA/sham, p<0.05, ANOVA.) C. Lung sections stained with hematoxylin and eosin are shown. (Original magnification, 200X; arrows in inset indicate eosinophils.) D. Airways responsiveness was measured in tracheotomized animals exposed to increasing doses of methacholine (N=3 mice per group, mean±SEM, *different from wild type OVA/RV mice, p<0.05, two-way ANOVA.) E–G. C57BL/6 wild-type mice were exposed to either PBS or OVA before treatment with IgG or anti-TLR2 and infection with RV or sham control. E. Lung neutrophils and eosinophils were assessed by flow cytometry, gating on high-complexity, CD45+, Gr1+, Siglec F+ cells. Whole lung mRNA expression of CXCL1 (G) and CCL24 (H) was measured by qPCR. (N=3 mice per group), mean±SEM, *different from IgG, †different from OVA/sham, p<0.05, ANOVA.)

Article Snippet: Selected cultures were also incubated with 50 ng/ml IL-4 (Peprotech), 30 μg/ml monoclonal anti-TLR2 neutralizing antibody (clone T2.5, eBioscience, San Diego, CA) or isotype control antibody Animals C57BL/6 mice WT and TLR2−/− (B6.129-Tlr2 tm1Kir /J TLR2 KO) were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Infection, Staining, Flow Cytometry, Expressing

Journal: The Journal of allergy and clinical immunology

Article Title: TLR2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation

doi: 10.1016/j.jaci.2016.01.037

Figure Lengend Snippet: Following 13 Gy 137Cs-gamma-irradiation, wild type bone marrow was transferred into TLR2−/− and wild type mice and, conversely, TLR2−/− bone marrow was transferred into TLR2−/− and wild-type mice. Six-to-eight weeks after transfer, mice were sensitized and challenged with OVA and exposed to sham or RV. A. BAL fluid was harvested for cell counts. B. Airways responsiveness was assessed by exposure to increasing doses of inhaled methacholine. Total respiratory resistance was measured with a Buxco plethysmograph. (N=6, mean±SEM, *different from sham, p<0.05, one-way or two-way ANOVA, as applicable.)

Article Snippet: Selected cultures were also incubated with 50 ng/ml IL-4 (Peprotech), 30 μg/ml monoclonal anti-TLR2 neutralizing antibody (clone T2.5, eBioscience, San Diego, CA) or isotype control antibody Animals C57BL/6 mice WT and TLR2−/− (B6.129-Tlr2 tm1Kir /J TLR2 KO) were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Irradiation

Journal: The Journal of allergy and clinical immunology

Article Title: TLR2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation

doi: 10.1016/j.jaci.2016.01.037

Figure Lengend Snippet: Bone marrow cells from either wild type or TLR2−/− mice were cultured in L929 media for five days, incubated in the presence or absence of IL-4 overnight, pulsed with carboxyfluorescein diacetate succinimidyl ester (CFSE) and instilled intratracheally into TLR2−/− mice. A. Immunofluorescence images showing CFSE-pulsed cells in the airways (green=CFSE, red=TLR2, blue=DAPI; magnification, 200x). B, C. BAL cell counts, whole lung mRNA expression and airway histology from mice 24 h after sham or RV infection. Transfer of untreated macrophages from wild type mice to TLR2−/− mice was sufficient for RV-induced neutrophilic airway inflammation, and transfer of IL-4-treated wild-type macrophages was sufficient for RV-induced eosinophilic inflammation. (N=6–9; as pooled value from three separate experiments, each with three mice per group), mean±SEM, *different from transfer of TLR2−/− cells, †different from transfer of untreated wild type cells, p<0.05, ANOVA.) Transfer of untreated macrophages increased whole lung mRNA expression of the neutrophil chemoattractant CXCL1, whereas transfer of IL-4-treated macrophages increased expression of both CXCL1 and the eosinophil chemoattractant CCL24 (N=3, mean±SEM, *different from transfer of TLR2−/− cells, †different from transfer of untreated wild type cells, p<0.05, ANOVA.) C. Airway sections from mice described in (B) were stained with hematoxylin and eosin and anti-major basic protein (MBP). D. Effect of macrophage adoptive transfer on airway mucus, as demonstrated by PAS staining. E. Effect of macrophage transfer on airway cholinergic responsiveness. TLR2−/− mice treated with IL-4-treated wild-type or TLR2−/− macrophages and then infected with RV. (N=6, mean±SEM, *different from transfer of TLR2−/− cells, p<0.05, two-way ANOVA.)

Article Snippet: Selected cultures were also incubated with 50 ng/ml IL-4 (Peprotech), 30 μg/ml monoclonal anti-TLR2 neutralizing antibody (clone T2.5, eBioscience, San Diego, CA) or isotype control antibody Animals C57BL/6 mice WT and TLR2−/− (B6.129-Tlr2 tm1Kir /J TLR2 KO) were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Cell Culture, Incubation, Immunofluorescence, Expressing, Infection, Staining, Adoptive Transfer Assay

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Toll-like receptor 2 (TLR2) expression in human lymphoid tissue. Lymphoid tissue was labelled with monoclonal antibodies (mAbs) specific for TLR2, CD14 and CD1a using the immunoperoxidase method. Original magnification 100× (panels a, c and d); 400× (b). The box in panel (a) indicates the section magnified in panel (b). (A) Human lymph nodes labelled with mAbs specific for TLR2+ cells are located in the medullary cords (M) of the lymph nodes. Macrophages (CD14+) and dendritic cells (DCs) (CD1a+) localized to the same region as TLR2-positive cells TLR2 expression was weak to negative in follicles (F). (B) TLR2-expressing cells in human spleen are located predominantly at the splenic cords and at the interface between the white pulp (WP) and the red pulp (RP) or marginal zone (MZ), the same site as macrophages and DCs. (C) TLR2-positive cells in thymus are found predominantly at the medullary junction (MJ) with a limited number located at the cortex (C) and the medulla (M). Macrophages and DCs are scattered throughout the medulla, cortex and medullary junction. CD1a+ thymocytes are abundant in the cortex. (D) TLR2-positive cells in tonsil are localized in the medullary area, as are macrophages and DCs.

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing, Bioprocessing

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Toll-like receptor 2 (TLR2) expression on monocytes and dendritic cells (DCs) in human lymphoid tissue. TLR2-expressing cells were characterized for cell lineage in human tonsil using two-colour immunofluorescence confocal laser microscopy. TLR2-positive cells expressed markers for DCs (CD1a) and macrophages (CD68 and CD14), but did not express T-cell (CD3) or B-cell (CD20) markers. Green images (left column) are CD markers, red images (center column) are TLR2 and superimposed images are shown in right column. Magnification 630×. The frequency of TLR2+ cells in lymphoid tissue is greater than the number of macrophages or DCs in situ as both macrophages and DCs populate lymphoid tissues and therefore the number of TLR2+ cells should be no less than the sum of CD14+ and CD1a+ cells.

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing, Immunofluorescence, Microscopy, In Situ

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Toll-like receptor 1 (TLR1) expression on monocytes and dendritic cells (DCs) in lymphoid tissue. (A) Human tonsils labelled with monoclonal antibodies (mAbs) specific for TLR1 (a, b). TLR1-positive cells in tonsil localized to the medulla. Original magnification 200× (a) and 400× (b). The box in panel (a) indicates the region magnified in panel (b). (B) TLR1-expressing cells were characterized for cell lineage in human tonsil using two-colour immunofluorescence confocal laser microscopy. TLR1-positive cells expressed markers for DCs (CD1a) and macrophages (CD14), but did not express T-cell (CD3) or B-cell (CD20) markers. Green images (left column) are CD markers, red images (centre column) are TLR1 and superimposed images are shown in the right column. Original magnification 630×. An arrow denotes the CD14+ cell in the center of the panel. (C) TLR1 co-localizes with TLR2 in lymphoid tissue. TLR1 (green) and TLR2 (red) were labelled and detected as described above (Fig. 2) and superimposed to identify co-localization.

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing, Bioprocessing, Immunofluorescence, Microscopy

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Toll-like receptor 2 (TLR2) expression on epithelial cells in lymphoid tissue. (A) Immunoperoxidase staining of sections demonstrates that TLR2-positive cells are also distributed throughout the tonsillar crypt epithelium (TCE). Original magnification 100× (a) and 200× (b). (B) Double-immunofluorescence labelling of tonsil sections with cytokeratin (CK) 8, 14 and 10/13 (green; left panel of each row) and TLR2 (red; centre panel of each row) demonstrate co-localization of TLR2 with CK10/13, but not CK8 or CK14. Original magnification 630×. (C) Double-immunofluorescence labelling with endothelial cell marker Factor VIII (green; first panel) and TLR2 (red) illustrates a lack of co-localization (right panel).

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing, Immunoperoxidase Staining, Immunofluorescence, Marker

Journal: Frontiers in Immunology

Article Title: Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells

doi: 10.3389/fimmu.2020.564699

Figure Lengend Snippet: Sequences of primers used for RT-PCR analysis.

Article Snippet: Additionally, DCs were stained with antibodies for mouse TLR1 conjugated with Alexa Fluor 647 or TLR2 conjugated with phycoerythrin (PE) (e-Bioscience, San Diego, CA, USA).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells

doi: 10.3389/fimmu.2020.564699

Figure Lengend Snippet: Pam3CSK4 enhances TLR1 expression. (A) Total RNA was isolated from DCs and subjected to RT-PCR to determine TLR1 to TLR9 mRNA expression levels. Each TLR mRNA expression normalized with that of β-actin is presented as the percentage change ± standard deviation of three separate experiments against TLR1 mRNA expression, set as 100% by densitometric analysis. Asterisk (*) indicates statistical significance at p < 0.05 between the TLR1 and other TLRs groups. (B) DCs were stimulated with 10 μg/ml of Pam3CSK4 for 48 h and isolated total RNA was subjected to RT-PCR to determine TLR1 or TLR2 mRNA expression levels. Left , a representative TLR1 and TLR2 mRNA expression determined by RT-PCR. Right , TLR1 and TLR2 mRNA expression normalized with that of β-actin is presented as the percentage change ± standard deviation of three separate experiments against non-treated TLR1 mRNA expression, set as 100% by densitometric analysis. Asterisk (*) indicates statistical significance at p < 0.05 between the non-treatment and treatment groups. (C) After stimulation with Pam3CSK4 at 10 μg/ml for 48 h, DCs were stained with antibodies for TLR1 conjugated with Alexa Fluor 647 or TLR2 conjugated with PE, and then subjected to flow cytometry. Mouse IgG2a conjugated with Alexa Fluor 647 were used as an isotype control (IC) antibody. The histograms are representative results from flow cytometry for TLR1 or TLR2 from each treatment group. The value given in each histogram is the percentage of TLR1- or TLR2-positive cells among 10,000 events of CD11c-positive cells.

Article Snippet: Additionally, DCs were stained with antibodies for mouse TLR1 conjugated with Alexa Fluor 647 or TLR2 conjugated with phycoerythrin (PE) (e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells

doi: 10.3389/fimmu.2020.564699

Figure Lengend Snippet: Pam3CSK4-induced BAFF expression is mediated through TLR2 and MyD88. (A) DCs from wild-type, TLR2- or MyD88-deficient mice were stimulated with Pam3CSK4 at 0-10 μg/ml for 72 h. The cells were then stained with antibodies for mouse CD11c conjugated with APC and BAFF conjugated with FITC, and subjected to flow cytometry. The histograms are representative results from flow cytometry for mBAFF, and the value in each histogram is the percentage of mBAFF-positive cells among 10,000 events of CD11c-positive cells. (B) Under the same culture condition, sBAFF levels in culture media were measured by ELISA. The values are the mean ± standard deviation of three separate experiments. Asterisk (*) indicates a significant difference at p < 0.05 between the non-treatment and treatment groups.

Article Snippet: Additionally, DCs were stained with antibodies for mouse TLR1 conjugated with Alexa Fluor 647 or TLR2 conjugated with phycoerythrin (PE) (e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells

doi: 10.3389/fimmu.2020.564699

Figure Lengend Snippet: NF-κB and Sp1 are major transcription factors involved in the Pam3CSK4-induced BAFF expression. (A) Nuclear extracts were prepared from DCs stimulated with 10 μg/ml of Pam3CSK4 for 0–8 h and incubated with 32 P-labeled oligonucleotides containing NF-κB, Sp1, or C/EBP consensus sequences. One picomole of an unlabeled probe (Cold) was used in a competition assay to confirm specific binding. Reaction products were separated on a polyacrylamide gel and visualized by autoradiography. (B) The 293/TLR1-TLR2 or 293/Null cells transiently transfected with the mouse BAFF promoter luciferase reporter construct together with pRL-TK vector were stimulated with 10 μg/ml of Pam3CSK4 for 24 h. (C, D) The cells were pre-treated with indicated inhibitor (BAY11-7082 for NF-κB inhibition or mithramycin A for Sp1 inhibition) for 1 h followed by stimulation with 10 μg/ml of Pam3CSK4 for an additional 24 h. DMSO (0.1%) was used as a vehicle control (VC) for each inhibitor. After the stimulation, cytosolic extracts were subjected to the luciferase activity assay. Values are the mean ± standard deviation of triplicates. Asterisk (*) indicates a significant difference at p < 0.05 between the non-treatment and treatment groups (B) , and between the Pam3CSK4 and other treatment groups (C, D) within the same cell-line, respectively. (E) The CFSE-labeled splenocytes were stimulated with the indicated amount of conditioned media of DC stimulated with Pam3CSK4, 10 µg/ml of Pam3CSK4, or 1 µg/ml of LPS for 72 h. B-cell proliferation was then determined by monitoring CFSE levels of the B220-positive cell population using flow cytometry. Values are the mean ± standard deviation of triplicates. Asterisk (*) indicates a significant difference at p < 0.05 between the non-treatment and treatment groups.

Article Snippet: Additionally, DCs were stained with antibodies for mouse TLR1 conjugated with Alexa Fluor 647 or TLR2 conjugated with phycoerythrin (PE) (e-Bioscience, San Diego, CA, USA).

Techniques: Expressing, Incubation, Labeling, Competitive Binding Assay, Binding Assay, Autoradiography, Transfection, Luciferase, Construct, Plasmid Preparation, Inhibition, Activity Assay, Standard Deviation, Flow Cytometry

Journal: Molecules

Article Title: Baicalein and Αlpha-Tocopherol Inhibit Toll-like Receptor Pathways in Cisplatin-Induced Nephrotoxicity

doi: 10.3390/molecules27072179

Figure Lengend Snippet: Effect of α-tocopherol and baicalein administration on TLR2 and TLR4 in control and treated groups. All data are expressed as mean ± SEM. Significant vs. control group refers to A, while significant vs. (cisplatin, -tocopherol, and baicalein) treatment groups refers to (B, C, D), respectively.

Article Snippet: Briefly, paraffin-embedded sections were subjected to antigen retrieval citrate buffer for 30 min. Then, the expression levels of Kelch-like ECH associatedprotein1 (Keap-1) (cat:sc-514914, Santa Cruz), toll-like receptor 4 (TLR4) (cat: A5258, AB clonal), and toll-like receptor 2 (TLR2) (cat: A11225, AB clonal) were detected at 1:100 dilution; biotinylated secondary antibody was added after washes, and the slides washed again with PBS.

Techniques:

Journal: Molecules

Article Title: Baicalein and Αlpha-Tocopherol Inhibit Toll-like Receptor Pathways in Cisplatin-Induced Nephrotoxicity

doi: 10.3390/molecules27072179

Figure Lengend Snippet: Microscopic pictures of adrenal sections immunostained against TLR2 from rat groups sacrificed after 10 days showing ( A ) positive cell score, significant vs. control group refers to A, while significant vs. (cisplatin, -tocopherol, and baicalein) treatment groups refers to (B, C, D), respectively, ( B ) control group, and ( C ) marked expression in positive brown tubular expression in Cisplatin group. ( D ) The positive brown tubular expression slightly decreased in renal sections from α-tocopherol group, ( E ) moderately decreased in renal sections from baicalein group, and ( F ) significantly decreased in renal sections from the combined treatment group. Black arrows indicate positive reaction. IHC counterstained with Mayer’s hematoxylin. High magnification ×: 400 bar 50.

Article Snippet: Briefly, paraffin-embedded sections were subjected to antigen retrieval citrate buffer for 30 min. Then, the expression levels of Kelch-like ECH associatedprotein1 (Keap-1) (cat:sc-514914, Santa Cruz), toll-like receptor 4 (TLR4) (cat: A5258, AB clonal), and toll-like receptor 2 (TLR2) (cat: A11225, AB clonal) were detected at 1:100 dilution; biotinylated secondary antibody was added after washes, and the slides washed again with PBS.

Techniques: Expressing

Journal: Molecules

Article Title: Baicalein and Αlpha-Tocopherol Inhibit Toll-like Receptor Pathways in Cisplatin-Induced Nephrotoxicity

doi: 10.3390/molecules27072179

Figure Lengend Snippet: Primers used for quantitative RT-PCR.

Article Snippet: Briefly, paraffin-embedded sections were subjected to antigen retrieval citrate buffer for 30 min. Then, the expression levels of Kelch-like ECH associatedprotein1 (Keap-1) (cat:sc-514914, Santa Cruz), toll-like receptor 4 (TLR4) (cat: A5258, AB clonal), and toll-like receptor 2 (TLR2) (cat: A11225, AB clonal) were detected at 1:100 dilution; biotinylated secondary antibody was added after washes, and the slides washed again with PBS.

Techniques: Sequencing

Journal: Journal of Reproduction & Infertility

Article Title: Lipopolysaccharide- and Lipoteichoic Acid-mediated Pro-inflammatory Cytokine Production and Modulation of TLR2, TLR4 and MyD88 Expression in Human Endometrial Cells

doi:

Figure Lengend Snippet: Assessment of TLR2, TLR4 and MyD88 expression by ESCs and WECs. A: Assessment of TLR2, TLR4 and MyD88 transcripts expression by ESCs and WECs using RT-PCR. B: Flow cytometric analysis of TLR2 and TLR4 expression by ESCs. C: Western blot analysis of TLR2 and TLR4 expression by ESCs. In RT-PCR and Western blot analyses, PBMC was used as positive control. Monocyte gate of PBMC served as positive area in flow cytometry. D: Immunofluorescent staining of TLR2 and TLR4 in ESCs. Monocytes and HL60 cells were used as positive cell controls for TLR2 and TLR4 immunofluorescent stainings, respectively. Reagent negative control (NC) slides received isotype- matched preimmune normal serum. Nuclei were counterstained with DAPI. P1-3: Three representative participants 1-3, ESCs1-3: Endometrial stromal cells from three representative participants, PBMC: Peripheral blood mononuclear cells, NAC: No amplification control

Article Snippet: For TLR2 staining, 5×10 5 living cells were incubated with 5 μg/ml of Alexa fluor 488-conjugated anti-TLR2 antibody (BD) diluted in PBS-BSA for 30 min . All incubations were performed on ice.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Flow Cytometry, Staining, Negative Control, Amplification

Journal: Journal of Reproduction & Infertility

Article Title: Lipopolysaccharide- and Lipoteichoic Acid-mediated Pro-inflammatory Cytokine Production and Modulation of TLR2, TLR4 and MyD88 Expression in Human Endometrial Cells

doi:

Figure Lengend Snippet: Effect of LTA on TLR2 and MyD88 gene expression in WECs. WECs were treated with LTA (10000 ng/ml ) for 8 hr and expression of TLR2 and MyD88 genes was evaluated by RT-PCR. Representative TLR2 and MyD88 PCR bands are shown at the bottom of each graph. AU: Arbitrary unit

Article Snippet: For TLR2 staining, 5×10 5 living cells were incubated with 5 μg/ml of Alexa fluor 488-conjugated anti-TLR2 antibody (BD) diluted in PBS-BSA for 30 min . All incubations were performed on ice.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Immunology

Article Title: Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells

doi: 10.1111/j.1365-2567.2010.03317.x

Figure Lengend Snippet: Gene-specific primers and probe combinations assayed by quantitative real-time polymerase chain reaction

Article Snippet: Flow cytometry Using a FACSCalibur (BD Biosciences, San Jose, CA) flow cytometer, the surface phenotype of cells was determined using four-colour staining with the following antibodies: fluorescein isothiocyanate-conjugated (-FITC) or allophycocyanin-conjugated CD11c (HL3), phycoerythrin-conjugated (-PE) CD80 (16-10A1) (BD-Pharmingen, Oxford, UK); TLR2-PE (6C2), TLR4/MD2-PE-Cy7 (MTS510) (eBioscience, San Diego, CA) and major histocompatibility class II (MHCII)-FITC (M5/114) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Sequencing

Journal: Immunology

Article Title: Differential regulation of Toll-like receptor signalling in spleen and Peyer's patch dendritic cells

doi: 10.1111/j.1365-2567.2010.03317.x

Figure Lengend Snippet: A greater percentage of spleen dendritic cells (DCs) express surface Toll-like receptor 2 (TLR2) and TLR4 than Peyer's patch (PP) DCs. (a) AutoMACS-separated CD11c+ cells from spleen and PP were gated on the expression of CD11c and MHCII as in Fig. 1(a). The expression of surface TLR2 and TLR4-MD2 was then determined on the double-positive population. Values in the upper right of the histograms indicate the mean fluorescence intensity of cells in the indicated gate, n = 3. Bars represent mean ± SEM of percentage of cells falling in the indicated gate of the CD11c+ MHCII+ population, n = 3. (b) autoMACS-separated, flow cytometrically sorted DCs were analysed for gene expression of tlr 1–9. Expression was determined as fold induction compared with β-actin housekeeper. There was no statistical difference between spleen and PP in the messenger RNA levels of any of the genes investigated. Bars represent mean ± SEM, n = 4 or n = 5. Significance determined by unpaired Student's t-test, ***P < 0·001, *P < 0·05.

Article Snippet: Flow cytometry Using a FACSCalibur (BD Biosciences, San Jose, CA) flow cytometer, the surface phenotype of cells was determined using four-colour staining with the following antibodies: fluorescein isothiocyanate-conjugated (-FITC) or allophycocyanin-conjugated CD11c (HL3), phycoerythrin-conjugated (-PE) CD80 (16-10A1) (BD-Pharmingen, Oxford, UK); TLR2-PE (6C2), TLR4/MD2-PE-Cy7 (MTS510) (eBioscience, San Diego, CA) and major histocompatibility class II (MHCII)-FITC (M5/114) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Fluorescence, Gene Expression