Journal: Journal of Innate Immunity

Article Title: TLRs1-10 Protein Expression in Circulating Human White Blood Cells during Bacterial and COVID-19 Infections

doi: 10.1159/000536593

Figure Lengend Snippet: Box plot representation of intracellular expression of TLR3 and TLR7 in neutrophils in septic shock and COVID-19 patients compared to healthy donors. The results are presented as mean fluorescence intensity (MFI). The main body of the boxplots show the 1st and 3rd quartiles. Horizontal lines in each box represent the median. * p < 0.05, ** p < 0.005.

Article Snippet: Within 3 h, blood cells were stained using 14 monoclonal antibodies coupled to a fluorescent dye for the 10 TLRs, CD14, CD16, and CD19 antigens: CD19-APC (clone SJ25C1, #17-0198-42); TLR1-PE (clone GD2.F4, #12-9911-42); TLR3-PE (clone TLR3.7, #12-9039-82), TLR10-PE (clone 3C10C5, #12-2909-42) from Invitrogen; CD16-APC-H7 (clone 3G8, #560195); CD14-PE-Cy7 (clone Mφ9, #562698); TLR4-BV421 (clone TF901, #564401); CD180-BV421 (clone G28-8, #743624) from BD Biosciences; TLR2-PerCP (clone 383936, #FAB2616C); TLR5-A488(clone 624915, #FAB6704G); TLR7-PerCP (clone 533707, #IC5875C); TLR8-A488 (clone 935166, #IC8999G); TLR9-A405 (clone 26C593R, #IC36583V) from R&D Systems and TLR6- FITC (clone TLR6.127, #ab72362) from Abcam.

Techniques: Expressing, Fluorescence

Journal: Scientific reports

Article Title: COVID-19 plasma exosomes promote proinflammatory immune responses in peripheral blood mononuclear cells.

doi: 10.1038/s41598-022-26457-8

Figure Lengend Snippet: Figure 6. Cytokine production and TLRs in response to plasma exosomes. (a) PBMCs were treated with the TLR3 inhibitor (10 µM) for 30 min or remained untreated, followed by stimulation with plasma exosomes (4 × 109 ml−1) for 16 h. Production of cytokines was determined by flow cytometry gated on CD4+, CD8+, and CD14+. COV exo, COVID-19 plasma exosome treatment; inh/cov, TLR3 inhibitor treated and COVID-19 plasma exosomes stimulated; non-COV, treatment with non-COVID plasma exosomes. *p < 0.05; ns, p > 0.05. ANOVA, equal variants. (b) CD4+ T cells, CD8+ T cells, and CD14+ monocytes were isolated from PBMC using MicroBeads, followed by treatment with plasma exosomes from COVID-19 patients upon admission (COV exo, 4 × 109 ml−1), non-COVID donors (non- COV exo, 4 × 109 ml−1), or poly(I:C) (5 µg ml−1) for 16 h at 37 °C. Expression of TLR3, TLR7, TLR8, and TLR9 was determined by flow cytometry gated on live cells. Isotype antibodies and no-antibody blanks were used in each flow cytometry assay. ctrl, medium only control. Error bars, ± SD; *p < 0.05; ns, p > 0.05; one-way ANOVA. Isotype antibody controls and blank controls were performed in parallel in flow cytometry.

Article Snippet: For flow cytometry, cells were stained using monoclonal antibodies: PE-conjugated IL-6 (Clone MQ2-13A5, BD Biosciences, Franklin Lakes, NJ), APC-conjugated TNF-α (Clone MAb11, BD Biosciences), PE-CF594-conjugated IL-8 (Clone G265-8, BD Biosciences), PE-CF594-conjugated IL-10 (Clone JES3-19F1, BD Biosciences), APC-R700-conjugated IL-17 (Clone N49-653, BD Biosciences), PerCP-Cy5.5-conjugated IFNγ (Clone B27, BD Biosciences), PerCP-Cy5.5-conjugated TGFβ (Clone TW4-9E7, BD Biosciences), PE-conjugated TLR3 (Clone TLR-104, Biolegend, San Diego, CA), PerCPconjugated TLR7 (Clone 533707, R&D systems), FITC anti-human CD288 (TLR8) antibody (Clone 16018A, Biolegend), and APC-conjugated TLR9 (Clone S16D, Biolegend).

Techniques: Clinical Proteomics, Flow Cytometry, Isolation, Expressing, Control