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: BMC Pulmonary Medicine

Article Title: By activating endothelium histone H4 mediates oleic acid-induced acute respiratory distress syndrome

doi: 10.1186/s12890-024-03334-w

Figure Lengend Snippet: TLR and calcium signaling involved in histone H4-mediated endothelium activation. After the MLVECs were challenged with histone H4 (15 mg/L) for 12 h, the changes in endothelial HS were evaluated by flow cytometry ( A ) while the VE-Cadherin in the cell membrane was measured by western blot ( B ). The vWF in the supernatant ( C, E ) and P-selectin translocation ( D, F ) were measured through ELISA. Prior to histone H4 challenge, the cells were pretreated for one hour with a blocking antibody (10 mg/L) against TLRs (TLR1, TLR2, TLR4, or TLR6) and the calcium chelator EGTA-AM (25, 50 or 100 µM). Data are presented as mean ± SD ( n = 6). The results for flow-cytometry and western blot are representative of three similar samples. * p < 0.05, ** p < 0.01 compared to the control group; # p < 0.05, ## p < 0.01 compared to the H4 group

Article Snippet: The reagents used in this study included: oleic acid (OA) and Histopaque (Sigma-Aldrich St. Louis, MO, USA); histone H4 (Millipore, Billerica, MA, USA); antibodies for P-selectin, sodium-potassium ATPase, CD31 (PECAM-1), and Ly6G (Abcam, Cambridge, MA, USA); an antibody for Cadherin-5 (BD Transduction Laboratories, CA, USA); an antibody for heparan sulfate (HS) (Bioss, Woburn, MA, USA); blocking antibodies against P-selectin, TLR4 (HTA125), TLR2 (TL2.1), and TLR1 (GD2.F4) (eBioscience, San Diego, CA, USA); a blocking antibody against TLR6 (TLR6.127) (Abcam, Cambridge, MA, USA); enzyme-linked immunosorbent assay (ELISA) kits for histone H4 and von Willebrand factor (vWF) (Cusabio Biotech, Wuhan, China); and 1,2-bis(2-aminophenoxy) ethane N, N,N’,N’-tetraacetic acid acetoxymethyl ester (EGTA-AM) (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Activation Assay, Flow Cytometry, Membrane, Western Blot, Translocation Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Vital NETosis vs. suicidal NETosis during normal pregnancy and preeclampsia

doi: 10.3389/fcell.2022.1099038

Figure Lengend Snippet: Comparisons of the percentages of total NETs (A) and citrullinated NETs (B) formed after stimulation of healthy volunteer neutrophils with plasma from women delivering a normal pregnancy (NP) or preeclampsia (PE) without inhibition and with inhibition by anti-TLR2 and TLR4 blocking antibodies. (A) NP without anti-TLR2 and TLR4 blocking antibodies 28.40% [26.06; 35.56]; NP with anti-TLR2 and TLR4 blocking antibodies 12.57% [9.59; 32.58]. PE without anti-TLR2 and TLR4 blocking antibodies 32.79% [30.17; 42.70]; PE with anti-TLR2 and TLR4 blocking antibodies 25.05% [14.10; 31.06]. (B) NP without anti-TLR2 and TLR4 blocking antibodies 16.77% [11.90; 19.65], NP with anti-TLR2 and TLR4 blocking antibodies 5.96% [1.85; 11.03]. PE without anti-TLR2 and TLR4 blocking antibodies 15.23% [9.97; 24.20]; PE with anti-TLR2 and TLR4 blocking antibodies 9.07% [4.92; 14.13]. (C) NP without anti-TLR2 and TLR4 blocking antibodies 12.80% [9.05; 18.23], NP with anti-TLR2 and TLR4 blocking antibodies 8.13% [6.55; 18.43]. PE without anti-TLR2 and TLR4 blocking antibodies 20.26% [14.87; 21.47]; PE with anti-TLR2 and TLR4 blocking antibodies 9.73% [4.79; 22.69]. Anti TLRs+: with inhibition by anti-TLR2 and TLR4 blocking antibodies. Results are represented with their medians and interquartile ranges [Q1; Q3]. The Bonferroni correction with a cut-off at 0.025 was applied to take into account the multiplicity of tests. ** p < 0.01, *** p < 0.001.

Article Snippet: As vital NETosis mainly passes through the TLR2 and four pathway ( ; ; ; ), we also performed the same experiments with anti-TLR2 and anti-TLR4 blocking antibodies (ANTI-HU CD284 HTA125 and ANTI-HU CD282 TL2.1 FG, Life Technologies SAS) at 5 μg/mL for 1 h at 37°C, 5% CO2.

Techniques: Inhibition, Blocking Assay