Journal: Frontiers in Medicine

Article Title: Toll-like receptor 4 and Syk kinase shape dendritic cell-induced immune activation to major house dust mite allergens

doi: 10.3389/fmed.2023.1105538

Figure Lengend Snippet: The role of TLR4 in HDM-induced moDC activation. (A) A HEK-293/TLR4 reporter cell line and the parental HEK-293 control cell line were incubated for 1 h at 37°C with either TLR4-blocking antibody or the respective isotype control antibody. Subsequently, cultures were exposed for 24 h to 10 ng/mL LPS or 10 μg/mL HDM extract, after which the culture supernatants were collected and screened for by ELISA for IL-8, that is expressed upon TLR4 engagement. The measured data are shown relative to levels obtained from LPS stimulation. The data are presented as the mean ± SD ( n = 3–4 donors). (B) MoDCs were incubated for 1 h at 37°C with either TLR4-blocking antibody or the respective isotype controls and subsequently exposed for 24 h to 10 ng/mL LPS or 50 μg/mL HDM extract. Culture supernatants were collected and screened for the cytokines IL-6, IL-10, and IL-12p70 by ELISA. The measured data are shown relative to levels obtained from untreated (control group) cells stimulated with HDM extract. The data are presented as the mean ± SD ( n = 5 donors). ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (RM-ANOVA analysis with Tukey’s multiple comparison test).

Article Snippet: After seeding the cultures into 96-well flat-bottom culture plates (Corning) and letting them adhere for 24 h, the cells were treated for 1 h with 30 μg/mL of either anti-human TLR4/MD-2 (clone 7E3, Hycult Biotech) or mouse IgG1 control antibody (Invivogen) prior to stimulation with 10 μg/mL HDM extract or LPS as a positive control.

Techniques: Activation Assay, Control, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Comparison