inducible nfb ap1 secreted alkaline phosphatase seap reporter  (InvivoGen)

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs activate the NF-κB/AP-1 pathways in human reporter macrophages via TLR2 signaling. A dTHP1-XBlue cells were treated with Ef -EVs (100 − 50,000 EVs/cell). LPS (100 ng/mL) was used as a positive control. B dTHP1-XBlue cells were treated with EVs derived from clinical E. faecalis bloodstream isolates (1000-10,000 EVs/cell). LPS (100 ng/mL) and Pam 3 CSK 4 (100 ng/mL) were used as positive controls. C-E) dTHP1-XBlue cells were pretreated with increasing concentrations (from 0.1 to 5 µg/mL) of anti-hTLR2-IgA mAb (dark pink) or human IgA2 control mAb (light pink) for 1 h. Subsequently, cells were treated with either (C) EVs (7000 EVs/cell), (D) Pam 3 CSK 4 (TLR2 ligand, 1 ng/mL), or (E) LPS (TLR4 ligand, 1 ng/mL) in the presence of antibodies for 24 h. Cells treated with cell culture medium supplemented only with Ef -EVs (7000 EVs/cell) in (C, blue), Pam 3 CSK 4 (1 ng/mL) in (D, dark blue), or LPS (1 ng/mL) in (E, red) were used as positive activating controls while cells treated with cell culture medium supplemented alone ( C - E , light green) were used as negative controls. NF-κB/AP-1 activation was measured as the activity of secreted SEAP and expressed as a normalized value relative to the positive controls. Data are shown as means ± SD of three independent experiments ( N = 3, n ≥ 3) and analyzed either by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test in (A and B) or by two-way analysis of variance (ANOVA) followed by Šídák multiple comparison post hoc test in ( C - E )

Article Snippet: Upon NF-κB/AP-1 and/or IL-8 activation via TLR2 stimulation, HEK-DualTM hTLR2 cells secrete SEAP and Lucia luciferase, which can be quantified using the QUANTI-BlueTM solution and the QUANTI-LucTM (cat. no. rep-qlc1, Invivogen), respectively.

Techniques: Positive Control, Derivative Assay, Control, Cell Culture, Activation Assay, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: TLR2 controls EV-induced immune activation but does not function as an endocytic receptor for EV uptake. dTHP1-XBlue cells were pretreated with anti-hTLR2-IgA mAb (1 µg/mL) or human IgA2 control mAb (1 µg/mL) for 1 h. Cells were then treated with DiI-labeled Ef -EVs (7000 EVs/cell) in the presence of antibodies for 4 and 24 h. Cells incubated in cell culture medium alone were used as negative controls. Cells treated with cell culture medium supplemented with DiI-labeled Ef -EVs (7000 EVs/cell) were used as positive activating controls. For EV internalization assays, mean fluorescence intensity (B and C, upper panel) and EV-positive cells (B and C, lower panel) were quantified after 4 ( A and B ) and 24 h ( A and C ) of EV treatment by measuring fluorescence intensity associated with DiI-labeled Ef -EVs on the PE channel. NF-κB/AP-1 activation in D was measured after 24 h of EV incubation as the activity of secreted SEAP and expressed normalized to the positive controls. Quantitative results are presented as mean ± SD ( N = 3, n = 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: Upon NF-κB/AP-1 and/or IL-8 activation via TLR2 stimulation, HEK-DualTM hTLR2 cells secrete SEAP and Lucia luciferase, which can be quantified using the QUANTI-BlueTM solution and the QUANTI-LucTM (cat. no. rep-qlc1, Invivogen), respectively.

Techniques: Activation Assay, Control, Labeling, Incubation, Cell Culture, Fluorescence, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: TLR2 engagement by Pam 3 CSK 4 -SUVs triggers inflammatory activation but does not enhance SUV internalization. A - C ) dTHP1-XBlue cells were treated with Pam 3 CSK 4 -SUVs (6 µM) containing increasing amounts of Pam 3 CSK 4 (from 0 to 0.4 mol% total SUV lipid composition) for 18 h. Cells incubated in cell culture medium alone were used as negative controls. Cells treated with cell culture medium containing Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.4 mol% total SUV lipid composition) were used as positive activating controls. D - F ) dTHP1-XBlue cells were pretreated with anti-hTLR2-IgA mAb (1 µg/mL) or human IgA2 control mAb (1 µg/mL) for 1 h. Cells were then treated with Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.04 mol% total SUV lipid composition) in the presence of antibodies for 18 h. Cells treated with cell culture medium containing Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.04 mol% total SUV lipid composition) were used as positive activating controls. For both experiments, cells incubated in cell culture medium alone were used as negative controls. Mean fluorescence intensity (B and E, upper panel) and SUV-positive cells (B and E, lower panel) were quantified after 18 h of treatment by measuring fluorescence intensity associated with SUVs on the APC channel. In C and F, NF-κB/AP-1 activation was measured after 18 h of SUV treatment as the activity of secreted SEAP and expressed normalized to the positive controls. Quantitative results are shown as mean ± SD ( N = 3, n = 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: Upon NF-κB/AP-1 and/or IL-8 activation via TLR2 stimulation, HEK-DualTM hTLR2 cells secrete SEAP and Lucia luciferase, which can be quantified using the QUANTI-BlueTM solution and the QUANTI-LucTM (cat. no. rep-qlc1, Invivogen), respectively.

Techniques: Activation Assay, Incubation, Cell Culture, Control, Fluorescence, Activity Assay, Comparison

Journal: Journal of Medicinal Chemistry

Article Title: Probing Immune Signatures of Conjugated Pattern Recognition Receptor Ligands Identifies Chimeras with Potent Adjuvant and Antitumor Activities

doi: 10.1021/acs.jmedchem.6c00372

Figure Lengend Snippet: Receptor-specific activities of individual PRR ligands and conjugated PRR ligands. HEK-Blue NOD2, TLR2, TLR7, TLR4, or HEK-Lucia RIG-I cells were treated with the compounds (1 or 10 μM as indicated) and the corresponding positive controls (1 μM MDP for NOD2, 100 nM Pam3CSK4 for TLR2, 1 μM imiquimod for TLR7, 100 ng/mL LPS for TLR4, and 100 ng/mL Poly­(I/C) for RIG-I) for 18 h. The activities are shown relative to the vehicle-treated control (0.1% DMSO). Data are mean ± SEM of three independent experiments. EC 50 values were determined in HEK-Blue NOD2, TLR2, and TLR7 cells in at least three independent experiments with eight concentrations (1 nM to 10 μM for NOD2 cells, and 0.1 nM to 1 μM for TLR2 and TLR7 cells).

Article Snippet: HEK-Blue NOD2 (Cat. code: hkb-hnod2), TLR2 (Cat. code: hkb-htlr2), TLR4 (Cat. code: hkb-htlr4), and TLR7 (Cat. code: hkb-htlr7) cell lines (Invivogen, San Diego, CA) are derived from HEK293 cells by cotransfection of hNOD2, hTLR2, hTLR4, or hTLR7 genes, respectively, and a nuclear factor-κB (NF-κB)-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene.

Techniques: Control