hek 293 cells invivogen hkb htlr2 human tlr4  (InvivoGen)

Journal: Frontiers in cellular and infection microbiology

Article Title: Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination.

doi: 10.3389/fcimb.2016.00072

Figure Lengend Snippet: FIGURE 2 | M. leprae stimulates the production of pro-inflammatory cytokines in human macrophages partly through TLR4 activation. Macrophages differentiated from isolated PBMC were treated with neutralizing antibody for TLR4 (N.A.TLR4) for 2 h followed by incubation for 24 h with killed M. leprae. Supernatants were collected and ELISAs for pro-inflammatory cytokines TNF-α, IL-6, and CXCL10 were performed. Graphs (A–C) are derived from n = 4 non-BCG-vaccinated healthy volunteers (white squares) and n = 4 BCG-vaccinated healthy volunteers (black squares). Wilcoxon signed-rank tests were performed for each cytokine and showed significant reductions in cytokine production by anti-TLR4 neutralizing antibody. Median reduction in pg/ml for TNF-α = 38.34 (P < 0.01), IL-6 = 29.49 (P < 0.01) and CXCL10 = 13.19 (P < 0.01).

Article Snippet: HEK 293 Blue human TLR4 (HEK-Blue-hTLR4) cells were purchased from InvivoGen (San Diego, USA).

Techniques: Activation Assay, Isolation, Incubation, Derivative Assay

Journal: Frontiers in cellular and infection microbiology

Article Title: Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination.

doi: 10.3389/fcimb.2016.00072

Figure Lengend Snippet: FIGURE 1 | M. leprae signals through TLR4. Human embryonic kidney (HEK) 293-Blue cell line expressing human TLR4, MD2/CD14 co-receptor genes and a secreted embryonic alkaline phosphatase (SEAP) reporter gene, was stimulated with increasing concentrations of killed whole M. leprae for 24 h with or without a monoclonal TLR4 neutralizing antibody (N.A.TLR4). The levels of alkaline phosphatase in the supernatants were detected in medium by reading the OD at 630 nm (MOI = Multiplicity of infection). ***p ≤0.0001 (student’s t-test) compared to unstimulated control.

Article Snippet: HEK 293 Blue human TLR4 (HEK-Blue-hTLR4) cells were purchased from InvivoGen (San Diego, USA).

Techniques: Expressing, Infection, Control

Journal: Frontiers in cellular and infection microbiology

Article Title: Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination.

doi: 10.3389/fcimb.2016.00072

Figure Lengend Snippet: FIGURE 3 | TLR4 expression in macrophages is modulated in BCG-vaccinated and non-BCG vaccinated healthy volunteers after exposure to killed M. leprae. (A) Histograms showing up-regulation of TLR4 expression in macrophages from a non-BCG vaccinated volunteer (upper histogram) and down-regulation of TLR4 expression in macrophages from a BCG-vaccinated healthy volunteer (lower histogram) after incubation with killed M. leprae for 18 h. (B) Percentage change of Median Fluorescence Intensity (MFI) indicating expression of TLR4 compared to unstimulated control (medium) after incubation of macrophages with increasing concentrations of killed M. leprae for 18 h. Using multicolor flow cytometry, macrophages were gated as CD68+ cell population. The plot shows the total % change in TLR4 expression compared to controls in macrophages from non-BCG vaccinated (black boxes) and BCG-vaccinated (gray boxes) healthy volunteers, after incubation with increasing MOI of killed M. leprae. (C) Subgroup analysis of percentage change of TLR4 MFI for the CD16+CD68+ macrophage population. Graphs summarize (n = 8) experiments with a total of 14 non-BCG vaccinated healthy volunteers and 10 BCG-vaccinated healthy volunteers. Mann-Whitney tests were used to compare the non-BCG vaccinated with the BCG-vaccinated donors. *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001, ****p < 0.0001.

Article Snippet: HEK 293 Blue human TLR4 (HEK-Blue-hTLR4) cells were purchased from InvivoGen (San Diego, USA).

Techniques: Expressing, Incubation, Fluorescence, Control, Cytometry, MANN-WHITNEY

Journal: Frontiers in cellular and infection microbiology

Article Title: Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination.

doi: 10.3389/fcimb.2016.00072

Figure Lengend Snippet: FIGURE 4 | Exposure to live BCG reverses the modulation of TLR4 expression by M. leprae in macrophages from non-BCG vaccinated healthy volunteers. Percentage change of Median Fluorescence Intensity (MFI) indicating expression of TLR4 compared to unstimulated control (medium) after incubation of differentiated human macrophages derived from FACS-sorted monocytes with increasing concentrations of killed M. leprae or LPS for 18 h. Using multicolor flow cytometry, macrophages were gated as CD68+ cell population. The plot shows the total % change in TLR4 expression compared to control (dashed line) in macrophages without pre-treatment with BCG (black boxes) and with pre-treatment with BCG MOI = 10 for 18 h (gray boxes), following incubation with MOI = 5 and MOI = 10 of killed M. leprae or with LPS 100 ng/ml for 18 h. Macrophages showing up-regulation of TLR4 expression after incubation with LPS for 18 h were considered LPS-responsive (A), whereas macrophages showing down-regulation of TLR4 after incubation with LPS were considered non-LPS responsive (B). Graphs summarize a total of five non-BCG vaccinated healthy volunteers responsive to LPS (A) and a total of six non-BCG vaccinated healthy volunteers non-responsive to LPS (B). Wilcoxon signed-rank tests were used to compare the different treatments with unstimulated control (dashed line). *0.01 < p < 0.05; **0.001 < p < 0.01.

Article Snippet: HEK 293 Blue human TLR4 (HEK-Blue-hTLR4) cells were purchased from InvivoGen (San Diego, USA).

Techniques: Expressing, Fluorescence, Control, Incubation, Derivative Assay, Cytometry