anti human tlr4 blocking antibody  (InvivoGen)

Journal: Journal of Biological Methods

Article Title: Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling

doi: 10.14440/jbm.2023.408

Figure Lengend Snippet: AZ617 is a human-specific, TLR4-dependent agonist that activates NFκB. A. Stimulation of human PBMCs, but not mouse splenocytes, with AZ617 for 24 hours effected a concentration-dependent release of NFκB-related cytokines IFNγ, IL-1β, TNFα, and IL-6. Representative graphs generated from 4 independent experiments. B. AZ617 activity was reduced when human PBMCs were pre-treated with an anti-human TLR4 blocking reagent for one hour prior to stimulation. Representative graphs derived from 2 independent experiments. Unpaired t-test **p#x003C;0.01, ***p#x003C;0.001. Error bars represent standard error of the mean (SEM).

Article Snippet: For TLR4-dependency studies, human PBMCs were pre-incubated with 12.5 μg/mL anti-human TLR4 blocking antibody (Invivogen, mabg-htlr4) for 1 hour at 37ºC prior to stimulation with 50 ng/mL AZ617 for an additional 24 hours.

Techniques: Concentration Assay, Generated, Activity Assay, Blocking Assay, Derivative Assay

Journal: Frontiers in Veterinary Science

Article Title: Lipopolysaccharide-Activated Canine Platelets Upregulate High Mobility Group Box-1 via Toll-Like Receptor 4

doi: 10.3389/fvets.2021.674678

Figure Lengend Snippet: LPS-mediated HMGB1 expression is dependent on platelet TLR4. Washed platelets from eight dogs were treated with function blocking antibodies, an isotype control or vehicle control prior to activation with 10 μM ADP, 5 μg/ml LPS, 0.01 U/ml thrombin or LPS in the presence of ADP. HMGB1 surface expression, measured by flow cytometry, was expressed as (A) percent (%) positive cells or (B) median fluorescence intensity (MFI) fold change on a log 10 scale. TLR4 inhibition modulated LPS-mediated HMGB1 expression in platelets primed with ADP. Each line represents the median while the box represents the 25th and 75th percentile. Whiskers represent the range. One-way repeated measures ANOVA was used to assess the effect of TLR4 inhibition on HMGB1 expression separately in ADP, ADP+LPS or thrombin activated platelets, followed by post-hoc analysis by Dunnett's multiple comparisons. * p < 0.05, # p < 0.01.

Article Snippet: Briefly, washed platelets (1 × 10 7 cells/ml) were treated with a function blocking monoclonal mouse anti-human TLR4 (CD284) antibody (50 μg/ml, low endotoxin, clone HTA125, BioRad, Hercules, CA) for 30 min at room temperature prior to activation.

Techniques: Expressing, Blocking Assay, Activation Assay, Flow Cytometry, Fluorescence, Inhibition

Journal: BMC Pulmonary Medicine

Article Title: TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages

doi: 10.1186/s12890-017-0517-0

Figure Lengend Snippet: LPS upregulates TLR4 and NOX2 expression and ROS levels in THP-1 cells. a THP-1 cells or THP-1(A) cells were treated with or without LPS, incubated with TLR4-PE, and bound TLR4-PE levels were measured using flow cytometry as an indicator of TLR4 expression. Additionally, some THP-1(A) cells were infected with M. tuberculosis . Histograms of the results, with percentage of cells expressing high TLR4 indicated. b Bar graph of percentages from a . (*; P < 0.05 compared to its control). c Western blot of NOX2 expression in THP-1(A) cells with or without pretreatment with LPS, with or without subsequent M. tuberculosis infection. d Bar graph of band intensities from c . (*; P < 0.05). e Intracellular ROS levels were measured using flow cytometry. f Bar graph of ROS levels from e . [*; P < 0.05 compared to LPS(−)]

Article Snippet: PMA (#P1585) and lipopolysaccharide were obtained from Sigma-Aldrich (Shanghai, China), fluorescein-labeled antibody TLR4-PE from eBioscience (San Diego, USA), anti-human TLR4 blocking antibody HTA125 and anti-human NOX2 protein antibody from Santa Cruz Biotechnology (Santa Cruz, USA), and NOX2 inhibitor diphenyleneiodonium (DPI) from Sigma.

Techniques: Expressing, Incubation, Flow Cytometry, Infection, Western Blot

Journal: BMC Pulmonary Medicine

Article Title: TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages

doi: 10.1186/s12890-017-0517-0

Figure Lengend Snippet: Blocking TLR4 and NOX2 inhibits the phagocytosis of M. tuberculosis by THP-1 cells. a THP-1(A) cells were incubated with M. tuberculosis -FITC for various time periods (1–6 h) at 0 °C or 37 °C. THP-1(A) cells were isolated and identified by flow cytometry (top left), and amount of M. tuberculosis -FITC in those cells was also measured by flow cytometry (bottom left). Histogram of results (middle) with percentage of cells containing high levels of FITC indicated for the 37 °C group. Bar graph of percentages (right). (*; P < 0.05 compared to t = 1 h). b THP-1(A) cells treated without LPS, with LPS, or with LPS in combination with DPI (10 μM), DPI (20 μM), or HTA125 pretreatment were infected with M. tuberculosis -FITC, isolated, and analyzed by flow cytometry. The proportion of cells containing high levels of FITC is indicated (top). Bar graph of percentages (bottom). (*; P < 0.05). c THP-1(A) cells were incubated with various concentrations of LPS (0–1000 ng/mL) and subsequently infected with various concentrations of M. tuberculosis -FITC (1:1–1:100). The proportion of cells containing high levels of FITC was measured as in b . Bar graph of the results. (*; P < 0.05 compared to lowest concentration of LPS). (FSC; forward scatter, SSC; side scatter)

Article Snippet: PMA (#P1585) and lipopolysaccharide were obtained from Sigma-Aldrich (Shanghai, China), fluorescein-labeled antibody TLR4-PE from eBioscience (San Diego, USA), anti-human TLR4 blocking antibody HTA125 and anti-human NOX2 protein antibody from Santa Cruz Biotechnology (Santa Cruz, USA), and NOX2 inhibitor diphenyleneiodonium (DPI) from Sigma.

Techniques: Blocking Assay, Incubation, Isolation, Flow Cytometry, Infection, Concentration Assay

Journal: BMC Pulmonary Medicine

Article Title: TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages

doi: 10.1186/s12890-017-0517-0

Figure Lengend Snippet: Blocking TLR4 and NOX2 inhibits the killing of M. tuberculosis by THP-1 cells. a M. tuberculosis was incubated with various concentrations of FDA to achieve labeling. Percentage of bacteria labeled is indicated. b FDA labeled M. tuberculosis (left most scatter plot) was incubated with THP-1(A) cells for various time periods (0–60 min). THP-1(A) cells were lysed and total phagocytized M. tuberculosis and M. tuberculosis in serum were isolated. FDA levels in M. tuberculosis were measured by flow cytometry with percentage of M. tuberculosis with FDA removed indicated (left). Graph of FDA removal levels (right). (*; P < 0.05 compared to t = 0, **; P < 0.01 compared to t = 0). c THP-1(A) cells treated without LPS, with LPS, or with LPS in combination with DPI or HTA125 pretreatment were infected with M. tuberculosis -FDA at 0 °C or 37 °C. THP-1(A) cells were lysed and total phagocytized M. tuberculosis and M. tuberculosis in serum were analyzed by flow cytometry for FDA levels. The number of viable M. tuberculosis retaining FDA is indicated (left). Graph of MFI from left (right). (*; P < 0.05). d THP-1(A) cells were treated with various concentrations of LPS (0–10,000 ng/mL), then infected with M. tuberculosis -FDA at 0 °C or 37 °C. THP-1(A) cells were lysed and total phagocytized M. tuberculosis and M. tuberculosis in serum were analyzed by flow cytometry for FDA levels. The number of viable M. tuberculosis retaining FDA is indicated. (SSC; side scatter, MFI; median fluorescence intensity)

Article Snippet: PMA (#P1585) and lipopolysaccharide were obtained from Sigma-Aldrich (Shanghai, China), fluorescein-labeled antibody TLR4-PE from eBioscience (San Diego, USA), anti-human TLR4 blocking antibody HTA125 and anti-human NOX2 protein antibody from Santa Cruz Biotechnology (Santa Cruz, USA), and NOX2 inhibitor diphenyleneiodonium (DPI) from Sigma.

Techniques: Blocking Assay, Incubation, Labeling, Isolation, Flow Cytometry, Infection, Fluorescence

Journal: BMC Pulmonary Medicine

Article Title: TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages

doi: 10.1186/s12890-017-0517-0

Figure Lengend Snippet: LPS upregulates TLR4 and NOX2 expression and ROS levels in THP-1 cells. a THP-1 cells or THP-1(A) cells were treated with or without LPS, incubated with TLR4-PE, and bound TLR4-PE levels were measured using flow cytometry as an indicator of TLR4 expression. Additionally, some THP-1(A) cells were infected with M. tuberculosis . Histograms of the results, with percentage of cells expressing high TLR4 indicated. b Bar graph of percentages from a . (*; P < 0.05 compared to its control). c Western blot of NOX2 expression in THP-1(A) cells with or without pretreatment with LPS, with or without subsequent M. tuberculosis infection. d Bar graph of band intensities from c . (*; P < 0.05). e Intracellular ROS levels were measured using flow cytometry. f Bar graph of ROS levels from e . [*; P < 0.05 compared to LPS(−)]

Article Snippet: PMA (#P1585) and lipopolysaccharide were obtained from Sigma-Aldrich (Shanghai, China), fluorescein-labeled antibody TLR4-PE from eBioscience (San Diego, USA), anti-human TLR4 blocking antibody HTA125 and anti-human NOX2 protein antibody from Santa Cruz Biotechnology (Santa Cruz, USA), and NOX2 inhibitor diphenyleneiodonium (DPI) from Sigma.

Techniques: Expressing, Incubation, Flow Cytometry, Infection, Western Blot

Journal: BMC Pulmonary Medicine

Article Title: TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages

doi: 10.1186/s12890-017-0517-0

Figure Lengend Snippet: Blocking TLR4 and NOX2 inhibits the phagocytosis of M. tuberculosis by THP-1 cells. a THP-1(A) cells were incubated with M. tuberculosis -FITC for various time periods (1–6 h) at 0 °C or 37 °C. THP-1(A) cells were isolated and identified by flow cytometry (top left), and amount of M. tuberculosis -FITC in those cells was also measured by flow cytometry (bottom left). Histogram of results (middle) with percentage of cells containing high levels of FITC indicated for the 37 °C group. Bar graph of percentages (right). (*; P < 0.05 compared to t = 1 h). b THP-1(A) cells treated without LPS, with LPS, or with LPS in combination with DPI (10 μM), DPI (20 μM), or HTA125 pretreatment were infected with M. tuberculosis -FITC, isolated, and analyzed by flow cytometry. The proportion of cells containing high levels of FITC is indicated (top). Bar graph of percentages (bottom). (*; P < 0.05). c THP-1(A) cells were incubated with various concentrations of LPS (0–1000 ng/mL) and subsequently infected with various concentrations of M. tuberculosis -FITC (1:1–1:100). The proportion of cells containing high levels of FITC was measured as in b . Bar graph of the results. (*; P < 0.05 compared to lowest concentration of LPS). (FSC; forward scatter, SSC; side scatter)

Article Snippet: PMA (#P1585) and lipopolysaccharide were obtained from Sigma-Aldrich (Shanghai, China), fluorescein-labeled antibody TLR4-PE from eBioscience (San Diego, USA), anti-human TLR4 blocking antibody HTA125 and anti-human NOX2 protein antibody from Santa Cruz Biotechnology (Santa Cruz, USA), and NOX2 inhibitor diphenyleneiodonium (DPI) from Sigma.

Techniques: Blocking Assay, Incubation, Isolation, Flow Cytometry, Infection, Concentration Assay

Journal: BMC Pulmonary Medicine

Article Title: TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages

doi: 10.1186/s12890-017-0517-0

Figure Lengend Snippet: Blocking TLR4 and NOX2 inhibits the killing of M. tuberculosis by THP-1 cells. a M. tuberculosis was incubated with various concentrations of FDA to achieve labeling. Percentage of bacteria labeled is indicated. b FDA labeled M. tuberculosis (left most scatter plot) was incubated with THP-1(A) cells for various time periods (0–60 min). THP-1(A) cells were lysed and total phagocytized M. tuberculosis and M. tuberculosis in serum were isolated. FDA levels in M. tuberculosis were measured by flow cytometry with percentage of M. tuberculosis with FDA removed indicated (left). Graph of FDA removal levels (right). (*; P < 0.05 compared to t = 0, **; P < 0.01 compared to t = 0). c THP-1(A) cells treated without LPS, with LPS, or with LPS in combination with DPI or HTA125 pretreatment were infected with M. tuberculosis -FDA at 0 °C or 37 °C. THP-1(A) cells were lysed and total phagocytized M. tuberculosis and M. tuberculosis in serum were analyzed by flow cytometry for FDA levels. The number of viable M. tuberculosis retaining FDA is indicated (left). Graph of MFI from left (right). (*; P < 0.05). d THP-1(A) cells were treated with various concentrations of LPS (0–10,000 ng/mL), then infected with M. tuberculosis -FDA at 0 °C or 37 °C. THP-1(A) cells were lysed and total phagocytized M. tuberculosis and M. tuberculosis in serum were analyzed by flow cytometry for FDA levels. The number of viable M. tuberculosis retaining FDA is indicated. (SSC; side scatter, MFI; median fluorescence intensity)

Article Snippet: PMA (#P1585) and lipopolysaccharide were obtained from Sigma-Aldrich (Shanghai, China), fluorescein-labeled antibody TLR4-PE from eBioscience (San Diego, USA), anti-human TLR4 blocking antibody HTA125 and anti-human NOX2 protein antibody from Santa Cruz Biotechnology (Santa Cruz, USA), and NOX2 inhibitor diphenyleneiodonium (DPI) from Sigma.

Techniques: Blocking Assay, Incubation, Labeling, Isolation, Flow Cytometry, Infection, Fluorescence

Journal: Journal of Virology

Article Title: Increased Escherichia coli -Induced Interleukin-23 Production by CD16 + Monocytes Correlates with Systemic Immune Activation in Untreated HIV-1-Infected Individuals

doi: 10.1128/JVI.01767-13

Figure Lengend Snippet: Percentages of CD16+ monocytes expressing TLR4 and TLR2. Flow cytometry was used to evaluate the percentages of CD16+ monocytes expressing TLR4 (A) and TLR2 (C) as well as the expression levels of TLR4 (B) and TLR2 (D) within total PBMC from HIV-1-infected (n = 32) and uninfected (n = 29) subjects prior to in vitro culture (baseline). Expression levels are shown as mean fluorescent intensity (MFI). Horizontal lines indicate median values. The statistical significance of the difference between HIV-1-infected and uninfected subjects was calculated using a Mann-Whitney test.

Article Snippet: CD14 + monocytes were cultured at 2 × 10 6 cells/ml in CM and were preincubated with an anti-human TLR4 blocking antibody or an isotype control (both at 20 μg/ml; both from eBioscience, San Diego, CA) for 30 min at 37°C.

Techniques: Expressing, Flow Cytometry, Infection, In Vitro, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Increased Escherichia coli -Induced Interleukin-23 Production by CD16 + Monocytes Correlates with Systemic Immune Activation in Untreated HIV-1-Infected Individuals

doi: 10.1128/JVI.01767-13

Figure Lengend Snippet: Contribution of TLR4 signaling to IL-23 production by total PBMC and CD14+ monocytes. (A) Production of IL-23 by total PBMC from HIV-1-infected (n = 22) and uninfected (n = 20) subjects after stimulation with purified LPS from E. coli. Results are shown as net IL-23 production. Horizontal lines indicate median values. The statistical significance of the difference between HIV-1-infected and uninfected subjects was calculated using a Mann-Whitney test. (B) Production of IL-23 by CD14+ monocytes from uninfected controls (n = 4) stimulated with purified LPS or whole E. coli in the presence of an anti-TLR4 antibody or an isotype control. Lines connect data from the same donor. The statistical significance of the difference between the blocking-antibody and isotype control conditions was calculated using a paired t test.

Article Snippet: CD14 + monocytes were cultured at 2 × 10 6 cells/ml in CM and were preincubated with an anti-human TLR4 blocking antibody or an isotype control (both at 20 μg/ml; both from eBioscience, San Diego, CA) for 30 min at 37°C.

Techniques: Infection, Purification, MANN-WHITNEY, Blocking Assay