tlr4  (Bio-Rad)

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of IFI16 lies within its N-terminal region (A) qRT-PCR analysis for TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with IFI16 alone (10 μg/mL) or pre-incubated for 1 h with the indicated amounts of anti-IFI16 polyclonal antibodies directed against either the N- or C-terminal region of the protein. Values are normalized to GAPDH mRNA and plotted as fold of induction over untreated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SEM (∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of TNF-α and IL-8 determined by ELISA in the culture supernatants harvested from human macrophages stimulated for 24 h as described in (A). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analysis of IFI16 binding to immobilized TLR4. 500 nM of IFI16 diluted in running buffer, alone or pre-incubated for 1 h at RT with increasing concentrations (62.5–500 nM) of the anti-N-term-IFI16 antibody (red bars) or with 500 nM of anti-C-term antibody (green bar), were flowed over a TLR4/MD2-coated chip. Data are representative of two independent experiments, shown as the mean ± SEM (∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test).

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-293072: RRID: AB_10611320.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Incubation, Protein Concentration, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The PYRIN domain of IFI16 is involved in TLR4/MD2 binding and activation (A) Schematic representation of the full-length IFI16 (IFI16 FL ), the truncated forms IFI16ΔHINB and IFI16ΔPYD, and the single domains used in this study. Numbers represent the amino acid positions based on the NCBI Reference Sequence GenBank: NP_005522 . (B) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with or without the recombinant proteins described in A (111 nM). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Protein concentration of TNF-α and IL-8 evaluated by ELISA in supernatants derived from human macrophages stimulated for 24 h as described in the legend (B), with or without CLI-095 (TLR4 inhibitor, 5μM). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis of IFI16 mutants or domains binding to immobilized TLR4/MD2. After immobilization of TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of IFI16 FL , IFI16ΔHINB, IFI16ΔPYD, IFI16 PYD , HINA, or HINB domains (20–800 nM), diluted in running buffer, were injected over the immobilized complex. Data are representative of three different experiments, with the dissociation constant (K D ) value provided where applicable. In the sensogram for IFI16ΔPYD, the asterisk (∗) denotes the mass transport effect observed at the highest concentration (2 μM). (E) Human macrophages were stimulated for 1 h with IFI16 FL , IFI16 PYD , IFI16ΔPYD, or left untreated (25 μg/mL). Total cellular extracts were then subjected to immunoprecipitation using an anti-TLR4 monoclonal antibody. Immunoprecipitates (left, IP:TLR4) and whole-cell lysates (right, Input) were analyzed by immunoblotting using antibodies against N-term-IFI16, C-term-IFI16 or TLR4. β-actin protein expression served as a protein loading control. Data are representative of three independent experiments with similar results.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-293072: RRID: AB_10611320.

Techniques: Binding Assay, Activation Assay, Sequencing, Quantitative RT-PCR, Expressing, Recombinant, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Injection, Immunoprecipitation, Western Blot, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of the PYRIN domain is conserved among PYHIN family members and across species Equimolar concentrations (111 nM) of the PYDs from various human and mouse family members, as well as from the unrelated NLRP3 and ASC proteins, were used to stimulate human (A and B) or mouse (C) macrophages for 24 h, with or without CLI-095 (TLR4 inhibitor, 5 μM). (A) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h. Values are normalized to GAPDH mRNA and plotted as fold induction over untreated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001, ∗∗ p < 0.01; one-way ANOVA followed by Dunnett’s test). (B and C) Protein concentration of TNF-α and IL-8 in the supernatants were evaluated by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗∗∗ p < 0.001, ### p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis showing the binding of various PYDs to TLR4/MD2. The TLR4/MD2 complex was immobilized on a CM5 sensor chip surface, followed by the injection of increasing concentrations of the indicated PYDs (20–800 nM) in running buffer. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-293072: RRID: AB_10611320.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Protein Concentration, Enzyme-linked Immunosorbent Assay, Binding Assay, Injection

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Structural analysis of the interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 PYD using the Robetta software. (B) Molecular docking analysis of the IFI16 PYD (green) and the extracellular portion of TLR4 (blue, PDB: 3FXI ) using High Ambiguity Driven protein-protein DOCKing software. (C) Alignment of the primary sequence of PYHIN PYDs along with those of NLRP3 and ASC performed using MEGAX and ClustalW algorithm to identify PYHIN-specific amino acids with similar chemical properties. Green arrows identify amino acids conserved across the different proteins, whereas light blue arrows identify amino acids that are conserved only across the PYHIN family members, which were used in mutagenesis experiments (see Figure 5 ). The red arrow identifies an amino acid conserved in all the proteins, which was mutated and used as control. The six α helices of the PYDs with known structures are also indicated. (D) Identification of the IFI16 PYD residues (green) potentially involved in the interaction with TLR4 (Lys34, Lys64, and Lys86) obtained by integrating the alignment and molecular docking information.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-293072: RRID: AB_10611320.

Techniques: Software, Sequencing, Mutagenesis, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Three amino acids located within the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar residues identified in Figures 4 E and 4F, highlighting their positions within the IFI16 PYD and the specific amino acid substitutions used in the mutagenesis experiments. (B) Human macrophages were stimulated for 24 h with equimolar concentrations (111 nM) of the IFI16 mutants described in (A ) and the IFI16 PYD−TM , alongside the wild type IFI16 FL . The levels of TNF-α released in the culture supernatants were measured by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) SPR analysis assessing the binding of IFI16 FL and its mutants to immobilized TLR4/MD2 on a CM5 sensor chip. Increasing concentration of IFI16 FL or the mutants (20–800 nM), diluted in running buffer, were flowed over the immobilized complex. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-293072: RRID: AB_10611320.

Techniques: Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of IFI16 lies within its N-terminal region (A) qRT-PCR analysis for TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with IFI16 alone (10 μg/mL) or pre-incubated for 1 h with the indicated amounts of anti-IFI16 polyclonal antibodies directed against either the N- or C-terminal region of the protein. Values are normalized to GAPDH mRNA and plotted as fold of induction over untreated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SEM (∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of TNF-α and IL-8 determined by ELISA in the culture supernatants harvested from human macrophages stimulated for 24 h as described in (A). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analysis of IFI16 binding to immobilized TLR4. 500 nM of IFI16 diluted in running buffer, alone or pre-incubated for 1 h at RT with increasing concentrations (62.5–500 nM) of the anti-N-term-IFI16 antibody (red bars) or with 500 nM of anti-C-term antibody (green bar), were flowed over a TLR4/MD2-coated chip. Data are representative of two independent experiments, shown as the mean ± SEM (∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test).

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Incubation, Protein Concentration, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The PYRIN domain of IFI16 is involved in TLR4/MD2 binding and activation (A) Schematic representation of the full-length IFI16 (IFI16 FL ), the truncated forms IFI16ΔHINB and IFI16ΔPYD, and the single domains used in this study. Numbers represent the amino acid positions based on the NCBI Reference Sequence GenBank: NP_005522 . (B) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with or without the recombinant proteins described in A (111 nM). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Protein concentration of TNF-α and IL-8 evaluated by ELISA in supernatants derived from human macrophages stimulated for 24 h as described in the legend (B), with or without CLI-095 (TLR4 inhibitor, 5μM). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis of IFI16 mutants or domains binding to immobilized TLR4/MD2. After immobilization of TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of IFI16 FL , IFI16ΔHINB, IFI16ΔPYD, IFI16 PYD , HINA, or HINB domains (20–800 nM), diluted in running buffer, were injected over the immobilized complex. Data are representative of three different experiments, with the dissociation constant (K D ) value provided where applicable. In the sensogram for IFI16ΔPYD, the asterisk (∗) denotes the mass transport effect observed at the highest concentration (2 μM). (E) Human macrophages were stimulated for 1 h with IFI16 FL , IFI16 PYD , IFI16ΔPYD, or left untreated (25 μg/mL). Total cellular extracts were then subjected to immunoprecipitation using an anti-TLR4 monoclonal antibody. Immunoprecipitates (left, IP:TLR4) and whole-cell lysates (right, Input) were analyzed by immunoblotting using antibodies against N-term-IFI16, C-term-IFI16 or TLR4. β-actin protein expression served as a protein loading control. Data are representative of three independent experiments with similar results.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Binding Assay, Activation Assay, Sequencing, Quantitative RT-PCR, Expressing, Recombinant, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Injection, Immunoprecipitation, Western Blot, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of the PYRIN domain is conserved among PYHIN family members and across species Equimolar concentrations (111 nM) of the PYDs from various human and mouse family members, as well as from the unrelated NLRP3 and ASC proteins, were used to stimulate human (A and B) or mouse (C) macrophages for 24 h, with or without CLI-095 (TLR4 inhibitor, 5 μM). (A) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h. Values are normalized to GAPDH mRNA and plotted as fold induction over untreated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001, ∗∗ p < 0.01; one-way ANOVA followed by Dunnett’s test). (B and C) Protein concentration of TNF-α and IL-8 in the supernatants were evaluated by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗∗∗ p < 0.001, ### p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis showing the binding of various PYDs to TLR4/MD2. The TLR4/MD2 complex was immobilized on a CM5 sensor chip surface, followed by the injection of increasing concentrations of the indicated PYDs (20–800 nM) in running buffer. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Protein Concentration, Enzyme-linked Immunosorbent Assay, Binding Assay, Injection

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Structural analysis of the interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 PYD using the Robetta software. (B) Molecular docking analysis of the IFI16 PYD (green) and the extracellular portion of TLR4 (blue, PDB: 3FXI ) using High Ambiguity Driven protein-protein DOCKing software. (C) Alignment of the primary sequence of PYHIN PYDs along with those of NLRP3 and ASC performed using MEGAX and ClustalW algorithm to identify PYHIN-specific amino acids with similar chemical properties. Green arrows identify amino acids conserved across the different proteins, whereas light blue arrows identify amino acids that are conserved only across the PYHIN family members, which were used in mutagenesis experiments (see Figure 5 ). The red arrow identifies an amino acid conserved in all the proteins, which was mutated and used as control. The six α helices of the PYDs with known structures are also indicated. (D) Identification of the IFI16 PYD residues (green) potentially involved in the interaction with TLR4 (Lys34, Lys64, and Lys86) obtained by integrating the alignment and molecular docking information.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Software, Sequencing, Mutagenesis, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Three amino acids located within the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar residues identified in Figures 4 E and 4F, highlighting their positions within the IFI16 PYD and the specific amino acid substitutions used in the mutagenesis experiments. (B) Human macrophages were stimulated for 24 h with equimolar concentrations (111 nM) of the IFI16 mutants described in (A ) and the IFI16 PYD−TM , alongside the wild type IFI16 FL . The levels of TNF-α released in the culture supernatants were measured by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) SPR analysis assessing the binding of IFI16 FL and its mutants to immobilized TLR4/MD2 on a CM5 sensor chip. Increasing concentration of IFI16 FL or the mutants (20–800 nM), diluted in running buffer, were flowed over the immobilized complex. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

Techniques: Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: Antibodies and protocols for indirect immunofluorescence analysis.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Incubation

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on paraffin human skin sections. Representative TLR2 ( A , B ), TLR4 ( C , D ), TLR7 ( E , F ), and TLR9 ( G , H ) immunostainings in normal human skin paraffin sections. ( A , C , E , G ): samples harvested at 24 h; ( B , D , F , H ): samples harvested at 48 h. ( A – H ): control samples. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; DAPI: 4′, 6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Control, Membrane, Immunostaining

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on IL-4 and IL-13 incubated paraffin human skin sections. Representative TLR2 ( A – D ), TLR4 ( E – H ), TLR7 ( I – L ), and TLR9 ( M – P ) immunostainings in normal human skin paraffin sections. ( A , E , I , M , C , G , K , O ): samples harvested at 24 h; ( B , F , J , N , D , H , L , P ): samples harvested at 48 h. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; IL-4: interleukin 4; IL-13: interleukin 13; DAPI: 4′, 6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Incubation, Membrane, Immunostaining

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: TLR2, TLR4, TLR7, and TLR9 immunofluorescence analysis on IL-22 and IL-23 incubated paraffin human skin sections. Representative TLR2 ( A – D ), TLR4 ( E – H ), TLR7 ( I – L ), and TLR9 ( M – P ) immunostainings in normal human skin paraffin sections. ( A , E , I , M , C , G , K , O ): samples harvested at 24 h; ( B , F , J , N , D , H , L , P ): samples harvested at 48 h. Nuclei are counterstained with DAPI. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; TLR7: Toll-like receptor 7; TLR9: Toll-like receptor 9; IL-22: interleukin 22; IL-23: interleukin 23; DAPI: 4′,6-diamidino-2-phenylindoledihydrochloride. White dotted line indicates the basal membrane. White arrows indicate positive immunostaining—scale bars: 50 µm.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Incubation, Membrane, Immunostaining

Journal: Journal of Clinical Medicine

Article Title: Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin

doi: 10.3390/jcm12051941

Figure Lengend Snippet: Quantitative analysis of TLR2 and TLR 4 epidermal distribution after immunofluorescence analysis after 24 and 48 h of cytokine incubation. ( A ): TLR2; ( B ): TLR4. TLR2: Toll-like receptor 2; TLR4: Toll-like receptor 4; IL-4: interleukin 4; IL-13: interleukin 13; IL-22: interleukin 22; IL-23: interleukin 23. Statistical analysis was performed by Prism 9.0.0 via Kruskal–Wallis non-parametric analysis of variance, followed by Dunn’s post-hoc multiple comparison test. Differences were considered statistically significant when p < 0.05. * p < 0.05; *** p < 0.01.

Article Snippet: Monoclonal mouse anti-human TLR4 (Novus Bio) , , 1:300 1 h at 37 °C.

Techniques: Immunofluorescence, Incubation, Comparison

Journal: PLoS ONE

Article Title: Porphyromonas Gingivalis and E-coli Induce Different Cytokine Production Patterns in Pregnant Women

doi: 10.1371/journal.pone.0086355

Figure Lengend Snippet: Leukocytes were selected in the forward-sidescatter plot (fig. 1A) and copied to a sidescatter-CD14 plot. Monocytes (CD14 positive cells), granulocytes (CD14 negative cells with high SSC) and lymphocytes (CD14 negative cells with low SSC) were gated (fig. 1B). CD14 positive cells were copied to a TLR2/TLR4 plot. Using the isotype control sample, gates were set in the TLR2/TLR4 plot so that at least 99% of the isotype controls were negative for TLR2/TLR4 expression (fig. 1C). This gate was then used to identify the percentages of TLR4/TLR2 double positive, TLR2 single and TLR4 single positive monocytes as well as their mean fluorescence intensity (MFI), in the antibody incubated samples (fig. 1D).

Article Snippet: Immediately after sampling, 500 µl of whole blood was mixed with 500 µl of RPMI and incubated with PerCp–labeled mouse-anti-human-CD14 (clone TüK4; Invitrogen Corporation, Breda, The Netherlands) together with FITC-labeled mouse-anti-human-TLR2 (clone TL2.1; eBioscience, Breda, The Netherlands) and PE-labeled mouse-anti-human-TLR4 (clone HTA 125; eBiosciences), or with anti-CD14 together with TLR2 and TLR4 isotype controls for 30 minutes at room temperature (RT) in the dark.

Techniques: Expressing, Fluorescence, Incubation

Journal: PLoS ONE

Article Title: Porphyromonas Gingivalis and E-coli Induce Different Cytokine Production Patterns in Pregnant Women

doi: 10.1371/journal.pone.0086355

Figure Lengend Snippet: Expression of TLR2 and TLR4 on monocytes of pregnant (open squares) and non-pregnant women (black squares). A: Percentage of TLR2 positive monocytes (left graph) and mean fluorescent intensity of TLR2 staining of monocytes (right graph). B: Percentage of TLR4 positive monocytes (left graph) and mean fluorescent intensity of TLR4 staining of monocytes (right graph). *: significantly increased vs pregnant women (student’s T test, p<0.05).

Article Snippet: Immediately after sampling, 500 µl of whole blood was mixed with 500 µl of RPMI and incubated with PerCp–labeled mouse-anti-human-CD14 (clone TüK4; Invitrogen Corporation, Breda, The Netherlands) together with FITC-labeled mouse-anti-human-TLR2 (clone TL2.1; eBioscience, Breda, The Netherlands) and PE-labeled mouse-anti-human-TLR4 (clone HTA 125; eBiosciences), or with anti-CD14 together with TLR2 and TLR4 isotype controls for 30 minutes at room temperature (RT) in the dark.

Techniques: Expressing, Staining