tlr2  (Bio-Rad)

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Primer names for PCR reactions.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Identified SNP variants within the coding sequence of bovine tlr2 in HF and BS breeds animals.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Residue

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Genotype frequency of Bos taurus and Bos indicus cattle breeds for TLR2 selected candidate SNP rs68343167 (H326Q).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Location of identified SNPs. Gene model for TLR2 from assembly UMD 3.1.1 with annotated SNPs. Missense SNPs are located on the first track below the gene model, with synonymous SNPS on the second track. Reading from left to right, exons and coding sequence (CDS; thick line) are shown. All mutations occur in the second exon, which codes for ligand binding side.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Ligand Binding Assay

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: TLR2-Ligand-Dependent NF-κB Activity and CXCL8 Secretion by HEK 293 cells Expressing Bovine and Human TLR2 Sequence Variants. HEK 293 cells, harboring NF-κB-induced SEAP reporter genes were transfected with either empty pTracer vector (mock); bovine or human TLR2 CDS constructs, or a bovine: human chimera in pDuo-mcs. Cells were stimulated with either FSL-1 at 100 ng mL -1 (A, C) , Pam3CSK4 at 1 mg mL -1 (B, D) for 24 hrs. SEAP activity was measured by quantifying optical density of cell culture supernatant at 635 nm (A, B) . OD values were normalised against values for PMA stimulation at 200 ng mL -1 . Additionally, supernatants were assessed for CXCL8 concentration by ELISA (C, D) . Error bars represent standard deviation from the mean of triplicate technical replicates and are representative of three independent repeats. (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001) and are only shown in relation to bovine TLR2 and additionally between the human and the chimeric bov: hu TLR 2 receptor. All data analysed by two-way ANOVA with multiple comparisons using GraphPad Prism V8 (GraphPad Inc., USA).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Activity Assay, Expressing, Sequencing, Transfection, Plasmid Preparation, Construct, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: M. bovis BCG and M. tuberculosis -Dependent NF-κB Activity Secretion by HEK 293 cells Expressing Bovine and Human TLR2 Sequence Variants. HEK 293 cells, harbouring NF-κB-induced SEAP reporter genes were transfected with either empty pTracer vector (mock); bovine or human TLR2 CDS constructs, or a bovine: human chimera in pDUO-mcs. Cells were stimulated with either live M. bovis BCG (MOI of 10) (A) or M. tuberculosis H37Rv (MOI of 5) (B) for 24 h. SEAP activity was measured by quantifying optical density of cell culture supernatant at 635 nm. OD values were normalised against values for PMA stimulation at 200 ng/ml. Error bars represent standard deviation from the mean of triplicate technical replicates and are representative of three independent repeats. (**=p<0.01, ***=p<0.001, ****=p<0.0001) and are only shown in relation to bovine TLR2 and additionally between the human and the chimeric bov: hu TLR 2 receptor. All data analysed by two-way ANOVA with multiple comparisons using GraphPad Prism V8 (GraphPad Inc., USA).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Activity Assay, Expressing, Sequencing, Transfection, Plasmid Preparation, Construct, Cell Culture, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: CXCL8 production by bovine MØ in response to FSL-1, M. bovis BCG or MTB Ligand Stimulation. Bovine MØ of BS (heterozygous for TLR2 H326Q SNP) and HF MØ (homozygous for the wild-type TLR2 sequence) were stimulated with either FSL-1 at 100 ng mL -1 (A) , 19 kDa lipoprotein antigen (represents Rv3763 or Mb3789) (EMC microcollections, Germany) or infected with M. bovis BCG at MOI = 5 (B) for 24 hr. CXCL8 concentration in cell culture supernatant was assessed by ELISA. A total of n=8 animals per breed were stimulated with FSL-1 and n=6 animals per breed were infected with M. bovis BCG. Error bars represent standard deviation from the mean. Data was analysed with two-way ANOVA and Tukey’s post hoc comparison in GraphPad Prism V8 (GraphPad Inc., USA) (*=p<0.05, ***=p<0.001).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Infection, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Pre-incubation with directly labeled TLR2 or TLR4 antibodies does not impact on phagocytosis of carboxyfluorescein succinimidyl ester (CSFE)-labeled E. bovis by polymorphonuclear neutrophils (PMN). Isolated PMN (1 × 10 6 per sample; n = 3) were pre-treated with TLR2 and TLR4 antibodies for 30 min and exposed to carboxyfluorescein succinimidyl ester (CFSE)-labeled E. bovis sporozoites (2.5 µm, 30 min) at a 1:1 ratio for two hours for subsequent flow cytometric analysis. Pre-incubation of PMN with antibodies to bovine TLR2 and TLR4 did not seem to impact on the phagocytosis of CFSE-labeled E. bovis sporozoites. Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc., San Diego, CA, USA).

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Incubation, Labeling, Isolation, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: E. bovis increases TLR2 and TLR4 expression on PMN. PMN (1 × 10 6 per sample; n = 3) were incubated with TLR2 and TLR4 antibodies for 30 min prior to exposure to live E. bovis for two hours for subsequent Flow cytometry analyses (FACS). Incubation of PMN with E. bovis significantly increases TLR2 ( A ) and TLR4 ( B ) expression (**** p < 0.0001). Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc.). p -value notation; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Expressing, Incubation, Flow Cytometry, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: IL-8 production in PMN upon E. bovis exposure. Supernatants of PMN (1 × 10 6 per sample; n = 3) treated with TLR2 and TLR4 antibodies and exposed to live E. bovis sporozoites (1:1 ratio; 2 h) were assessed for the presence of IL-8 by ELISA analysis. TLR2-treated PMN exposed to sporozoites showed a significant increase in IL-8 production (* p < 0.05) when compared to PMN in media. Likewise, a significant increase in IL-8 production (** p < 0.01) was observed in the same experimental condition when compared to the respective control without exposure to E. bovis. Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc., San Diego, CA, USA). p -value notation; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Induction of Toll-like receptor (TLR)-dependent NF-κB activation by E. bovis sporozoites. In order to investigate the activation of TLRs in bovine PMN, we used HEK cells expressing either bovine TLR2 (A) or a combination of bovine TLR4/MD2 (B). Cells were exposed to different E. bovis sporozoite preparations: live, heat killed (HK) or antigen ( Eb Ag) for 24 h and assessed for activation of transcription factor NF-κB using a luciferase reporter assay. ( A ) HK sporozoites and Eb Ag induced substantial TLR2-dependent NF-κB activation compared to media alone (*** p < 0.0001, ** p < 0.01 and * p < 0.05, respectively). TLR2-induced NF-κB significantly increases when exposed to Eb Ag compared to live E. bovis ( p < 0.05). ( B ) HK sporozoites induced a significant NF-κB response when compared to media ( p < 0.05) and when compared to live parasitic stages ( p < 0.01). A significant increase in NF-κB response was observed in Eb Ag when compared to media ( p < 0.05). In both experiments, Pam 3 CSK 4 and Lipopolysaccharides (LPS) served as ligand controls for TLR2 and TLR4/MD2, respectively, and phorbol 12-myristate 13-acetate (PMA) was used as an NF-κB technical control (data not shown for clarity). Data are represented as the mean of 3 replicates ± SD and were analyzed using an unpaired Student’s t -test using GraphPad Prism V.8.4.3 (GraphPad Software Inc.).

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Activation Assay, Expressing, Luciferase, Reporter Assay, Control, Software

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Immunofluorescence analysis on bovine PMN activation of TLR2 and TLR 4 by E. bovis and concomitant neutrophil extracellular trap (NET) formation. PMN ( n = 3; 5 × 10 5 ) were exposed to vital E. bovis sporozoites (ratio 1:1) on poly- l -lysine-treated coverslips (120 min, 37 °C) and fixed for further antibody exposure (60 min) with anti-TLR2 ( C ) and anti-TLR4 ( D ) antibodies and anti-histone H1, H2A/H2B, H3, H4 antibody ( E , F ). Coverslips were mounted with ProLong Antifade containing DAPI ( A , B ) which was used for observation of PMN nuclei and NET extracellular DNA by fluorescence microscopy analysis. In both cases, expression of TLR2 and TLR4 was observed on the surface of bovine PMN (red) co-localized with NET-derived histones (green) and extracellular DNA (blue), as indicated by yellow arrows (( G , H )—overlay of images collected for nucleic acid, TLR2/TLR and histone staining). Co-localization of TLR-positive signals with early stages of NETosis are indicated by white arrows ( G , H ). TLR-positive signals without NETosis are indicated by orange arrows ( G , H ). Images were visualized by using an inverted Olympus IX81 ® epifluorescence microscope equipped with a digital camera (XM10 ® , Olympus, Tokyo, Japan). Scale bar magnitude: 20 µm.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Immunofluorescence, Activation Assay, Fluorescence, Microscopy, Expressing, Derivative Assay, Staining

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Antibodies.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Recombinant

Journal: Pathogens

Article Title: The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

doi: 10.3390/pathogens10020118

Figure Lengend Snippet: Antibodies.

Article Snippet: Alexa Fluor 647 conjugated anti-bovine TLR2 , Bio-Rad , HCA152A647 (clone AbD12538) , HuCal Fab.

Techniques: Recombinant