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thp1 dual ko tlr4 md (InvivoGen)

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InvivoGen thp1 dual ko tlr4 md
$a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of <t>TLR4</t> signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.$
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1) Product Images from "Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling"

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

Journal: Nature Communications

doi: 10.1038/s41467-024-53770-9

$a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of TLR4 signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.$
Figure Legend Snippet: a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of TLR4 signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.

Techniques Used: Cell Culture, Refractive Index, IF-cells, Concentration Assay, Control, Incubation, Generated

Figure Legend Snippet: Pharmacological parameter of LPS induced DMR at six selected time points in HEK293 TLR4/MD-2/CD14 reporter cells

Techniques Used:

a HEK293 TLR4/MD-2/CD14 reporter cells in suspension were stimulated with the indicated concentrations (ng/ml) of LPS from E. col i with or without 50 µM of the TLR4-antagonist TAK-242. HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with the indicated concentrations of actin and microtubule inhibitors ( b ) cytochalasin B, ( c ) latrunculin A or ( d ) nocodazole stimulated with LPS E. coli (1000 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. ( a – d , right panels) Values at 250 min are presented as mean + SEM and are normalized to LPS E. coli (1000 ng/ml) ( n = 3 biologically independent experiments). Two-tailed Student's t test ( a ) and one-way analysis of variance (ANOVA, Tukey’s post-test) ( b – d ). Source data are provided as a Source Data file.

Figure Legend Snippet: a HEK293 TLR4/MD-2/CD14 reporter cells in suspension were stimulated with the indicated concentrations (ng/ml) of LPS from E. col i with or without 50 µM of the TLR4-antagonist TAK-242. HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with the indicated concentrations of actin and microtubule inhibitors ( b ) cytochalasin B, ( c ) latrunculin A or ( d ) nocodazole stimulated with LPS E. coli (1000 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. ( a – d , right panels) Values at 250 min are presented as mean + SEM and are normalized to LPS E. coli (1000 ng/ml) ( n = 3 biologically independent experiments). Two-tailed Student's t test ( a ) and one-way analysis of variance (ANOVA, Tukey’s post-test) ( b – d ). Source data are provided as a Source Data file.

Techniques Used: Suspension, Two Tailed Test

THP-1 monocytes ( a ) or macrophages ( b ) were stimulated with increasing concentrations (ng/ml) of LPS E.coli and LPS S. minnesota . THP-1 KO-TLR4 monocytes ( c ) or macrophages ( d ) were stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . THP1-Dual TLR4/MD-2/CD14 (THP-1 Dual) ( e ) or THP1-Dual KO-TLR4/MD-2/CD14 (THP-1 Dual TLR4-KO) ( f ) cells stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . Heatmap of the top 100 significant up- or downregulated genes identified in HEK293 TLR4/MD-2/CD14 cells ( g ) or THP-1 macrophages ( h ) treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. The global transcriptional response induced by the LPS chemotypes showed no significant differences. n = 2 biologically independent experiments. Venn diagram for HEK293 TLR4/MD-2/CD14 cells ( i ) and THP-1 macrophages ( j ) indicating the number of significant (FDR < 0.05) differentially expressed genes and the overlap between each set of genes treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. k , l THP1 monocytes (k) or macrophages (l) were stimulated with increasing concentrations (ng/ml) Pam 3 CSK 4 or Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file.

Figure Legend Snippet: THP-1 monocytes ( a ) or macrophages ( b ) were stimulated with increasing concentrations (ng/ml) of LPS E.coli and LPS S. minnesota . THP-1 KO-TLR4 monocytes ( c ) or macrophages ( d ) were stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . THP1-Dual TLR4/MD-2/CD14 (THP-1 Dual) ( e ) or THP1-Dual KO-TLR4/MD-2/CD14 (THP-1 Dual TLR4-KO) ( f ) cells stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . Heatmap of the top 100 significant up- or downregulated genes identified in HEK293 TLR4/MD-2/CD14 cells ( g ) or THP-1 macrophages ( h ) treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. The global transcriptional response induced by the LPS chemotypes showed no significant differences. n = 2 biologically independent experiments. Venn diagram for HEK293 TLR4/MD-2/CD14 cells ( i ) and THP-1 macrophages ( j ) indicating the number of significant (FDR < 0.05) differentially expressed genes and the overlap between each set of genes treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. k , l THP1 monocytes (k) or macrophages (l) were stimulated with increasing concentrations (ng/ml) Pam 3 CSK 4 or Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file.

Techniques Used: Control, Incubation

a Primary monocytes isolated from PBMCs were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S. minnesota . b Primary monocytes isolated from PBMCs were preincubated with 50 µM of the TLR4 antagonist TAK-242 and stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S . minnesota . c Primary monocytes isolated from PBMCs stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 and Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments and donors. Source data are provided as a Source Data file.

Figure Legend Snippet: a Primary monocytes isolated from PBMCs were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S. minnesota . b Primary monocytes isolated from PBMCs were preincubated with 50 µM of the TLR4 antagonist TAK-242 and stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S . minnesota . c Primary monocytes isolated from PBMCs stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 and Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments and donors. Source data are provided as a Source Data file.

Techniques Used: Isolation

a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.

Figure Legend Snippet: a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.

Techniques Used: Immunofluorescence, Microscopy, Transfection, Construct

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Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of TLR4 signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Cell Culture, Refractive Index, IF-cells, Concentration Assay, Control, Incubation, Generated

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: Pharmacological parameter of LPS induced DMR at six selected time points in HEK293 TLR4/MD-2/CD14 reporter cells

Techniques:

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a HEK293 TLR4/MD-2/CD14 reporter cells in suspension were stimulated with the indicated concentrations (ng/ml) of LPS from E. col i with or without 50 µM of the TLR4-antagonist TAK-242. HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with the indicated concentrations of actin and microtubule inhibitors ( b ) cytochalasin B, ( c ) latrunculin A or ( d ) nocodazole stimulated with LPS E. coli (1000 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. ( a – d , right panels) Values at 250 min are presented as mean + SEM and are normalized to LPS E. coli (1000 ng/ml) ( n = 3 biologically independent experiments). Two-tailed Student's t test ( a ) and one-way analysis of variance (ANOVA, Tukey’s post-test) ( b – d ). Source data are provided as a Source Data file.

Techniques: Suspension, Two Tailed Test

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: THP-1 monocytes ( a ) or macrophages ( b ) were stimulated with increasing concentrations (ng/ml) of LPS E.coli and LPS S. minnesota . THP-1 KO-TLR4 monocytes ( c ) or macrophages ( d ) were stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . THP1-Dual TLR4/MD-2/CD14 (THP-1 Dual) ( e ) or THP1-Dual KO-TLR4/MD-2/CD14 (THP-1 Dual TLR4-KO) ( f ) cells stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . Heatmap of the top 100 significant up- or downregulated genes identified in HEK293 TLR4/MD-2/CD14 cells ( g ) or THP-1 macrophages ( h ) treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. The global transcriptional response induced by the LPS chemotypes showed no significant differences. n = 2 biologically independent experiments. Venn diagram for HEK293 TLR4/MD-2/CD14 cells ( i ) and THP-1 macrophages ( j ) indicating the number of significant (FDR < 0.05) differentially expressed genes and the overlap between each set of genes treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. k , l THP1 monocytes (k) or macrophages (l) were stimulated with increasing concentrations (ng/ml) Pam 3 CSK 4 or Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file.

Techniques: Control, Incubation

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a Primary monocytes isolated from PBMCs were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S. minnesota . b Primary monocytes isolated from PBMCs were preincubated with 50 µM of the TLR4 antagonist TAK-242 and stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S . minnesota . c Primary monocytes isolated from PBMCs stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 and Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments and donors. Source data are provided as a Source Data file.

Techniques: Isolation

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.

Techniques: Immunofluorescence, Microscopy, Transfection, Construct

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