htlr5ni  (InvivoGen)

Journal: iScience

Article Title: Flagellin in blood and Bifidobacterium pseudocatenulatum in gut are associated with live birth upon IVF-frozen embryo transfer

doi: 10.1016/j.isci.2025.111933

Figure Lengend Snippet:

Article Snippet: HEK-Dual™-hTLR5 reporter cell line , InvivoGen , Cat. #hkd-htlr5ni.

Techniques: Recombinant, Purification, LAL Assay, Sequencing, Software

Journal: Biomolecules

Article Title: Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin

doi: 10.3390/biom12050624

Figure Lengend Snippet: List of bacterial strains, cell lines, plasmids and primers.

Article Snippet: HEK-Blue TM hTLR5 , HEK 293 cell line expressing human TLR5, SEAP reporter , InvivoGen, Toulouse, France.

Techniques: Expressing, Mutagenesis, Plasmid Preparation

Journal: Biomolecules

Article Title: Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin

doi: 10.3390/biom12050624

Figure Lengend Snippet: ProA inhibits TLR5 activation by FlaA in HEK-Blue™ cells. HEK-Blue™ hTLR5 cells were seeded at a density of 2.52 × 10 4 cells/well. After adherence, they were inoculated with HEK-Blue™ Detection medium and 20 µL samples containing the indicated bacterial strains or proteins. PBS served as a negative or background control (black dots). After incubation at 37 °C and 5% CO 2 for 16 h, SEAP activity, and hence hTLR5 stimulation, were determined at OD 620 . Means of measurements are shown with ±SEM from three ( A , B ) or four ( C , D ) independent experiments. ( A ) HEK-Blue™ cells were treated with PBS or 1 µg/mL ProA (white squares) and different concentrations of purified FlaA, only leading to increasing hTLR5 activation in the negative control. ( B ) 30 ng/mL FlaA per well (white squares) were added to HEK-Blue™ cells, which were additionally treated with different ProA concentrations between 0.1 ng/mL and 5000 ng/mL. TLR5 stimulation was thereby inhibited in a concentration-dependent manner. ( C ) HEK-Blue™ cells were inoculated with the L. pneumophila Corby Δ flaA mutant (grey triangles). Co-incubation with purified flagellin reconstituted the lacking TLR5 stimulation of the eukaryotic cells (white squares). ( D ) L. pneumophila Corby Δ proA inoculation of HEK-Blue™ cells resulted in high hTLR5 activation (grey triangles), but was diminished by adding natively purified ProA in increasing concentrations (white squares).

Article Snippet: HEK-Blue TM hTLR5 , HEK 293 cell line expressing human TLR5, SEAP reporter , InvivoGen, Toulouse, France.

Techniques: Activation Assay, Control, Incubation, Activity Assay, Purification, Negative Control, Concentration Assay, Mutagenesis

Journal: Biomolecules

Article Title: Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin

doi: 10.3390/biom12050624

Figure Lengend Snippet: Specific TLR5 activation in HEK-Blue™ cells by different L. pneumophila strains. HEK-Blue™ hTLR5 cells at a density of 2.52 × 10 4 cells/well were incubated with 180 µL detection medium and 20 µL culture of the L. pneumophila Corby wild type (WT), the negative mutant strains Δ proA and Δ flaA or complementants Δ proA proA and Δ flaA flaA in a 96-well format. Prior to inoculation, bacterial strains were grown to stationary phase in YEB, and the cell count was adjusted to 10 8 CFU/mL. After incubation of the assay plate at 37 °C and 5% CO 2 for 16 h, TLR5-dependent NF-κB activation was measured by SEAP activity at OD 620 . The results are depicted in a scatter plot with means and reveal significantly diminished values in the case of the flaA -negative strain but even stronger induced TLR5 stimulation by the proA deletion mutant compared to the WT. Complementation strains of Δ proA and Δ flaA were able to completely reconstitute these phenotypes. Statistics were performed using a repeated measurement one-way ANOVA with Dunnett’s post hoc test for simple effect analysis. Significant results of ten biological replicates are indicated with asterisks (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001).

Article Snippet: HEK-Blue TM hTLR5 , HEK 293 cell line expressing human TLR5, SEAP reporter , InvivoGen, Toulouse, France.

Techniques: Activation Assay, Incubation, Mutagenesis, Cell Counting, Activity Assay

Journal: Biomolecules

Article Title: Zinc Metalloprotease ProA from Legionella pneumophila Inhibits the Pro-Inflammatory Host Response by Degradation of Bacterial Flagellin

doi: 10.3390/biom12050624

Figure Lengend Snippet: TLR5 activation in HEK-Blue™ cells by supernatants of infected lung tissue explants (HLTEs). Explanted human lung tissue specimens were infected with the L. pneumophila Corby WT, a proA -negative mutant Δ proA and a flaA -negative mutant Δ flaA . Loss of ProA or FlaA was reconstituted either by addition of purified proteins (1 µg/mL ProA and 100 ng/mL monomeric FlaA) ( A ) or use of the specific complementation strains Δ proA proA and Δ flaA flaA ( B ). Untreated tissue samples served as a negative control. Infected tissue pieces were weighed, and supernatants were isolated and incubated with HEK-Blue™ hTLR5 cells for 16 h at 37 °C and 5% CO 2 . Activation of the TLR5-NF-κB pathway was measured by SEAP activity at OD 620 and is shown in scatter plots with means. Compared to the L. pneumophila WT, a HEK-Blue™ Detection assay revealed significantly reduced TLR5 activation by Δ flaA -infected tissue supernatant and elevated stimulation with the Δ proA mutant strain. These effects were apparent after 2 h ( A ) and 24 h ( B ) post infection and were successfully restored by complementation with respective proteins or gene sequences. Significance of at least twelve replicates was evaluated by repeated measurement one-way ANOVA with Dunnett’s post hoc test for simple effect analysis (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001).

Article Snippet: HEK-Blue TM hTLR5 , HEK 293 cell line expressing human TLR5, SEAP reporter , InvivoGen, Toulouse, France.

Techniques: Activation Assay, Infection, Mutagenesis, Purification, Negative Control, Isolation, Incubation, Activity Assay, Detection Assay