tlr7 8 agonists  (InvivoGen)

Journal: Frontiers in Immunology

Article Title: De novo COVID-19-associated insulin resistance drives dysregulated neutrophil extracellular trap formation (NETosis) four months after infection

doi: 10.3389/fimmu.2026.1787799

Figure Lengend Snippet: Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification of TLR7/8 and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) CL075 (1μg/mL), (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.

Article Snippet: For NETosis, 1 x 10 6 cells were incubated for 30 minutes at 37 °C and 5% CO2 under basal conditions or using IL-6 (20ng/mL) and TNFα (2ng/mL), or TLR7/8 agonists (CL075 (1μg/mL), ImiQ (2μg/mL), and R848 (2μg/mL), ssRNA40 (1μg/mL) and ssRNA 41(1μg/mL) (Catalog No.tlrl-kit3hw3, InvivoGen).

Techniques: Flow Cytometry, Staining, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: Integrated analysis of sex differences in human dendritic cell, monocyte, and natural killer cell subsets

doi: 10.3389/fimmu.2026.1750775

Figure Lengend Snippet: Frequency and functions comparisons of DC subsets in females and males. (A) Representative flow cytometry gating strategy for the identification of human DC subsets. Home-made lineage markers included CD3, CD19, CD56. AXL + DC: lineage - HLA-DR + AXL + ; pDCs: lineage - HLA-DR + AXL - CD11c - CD123 + ; CD141 + DC: lineage - HLA-DR + AXL - CD123 - CD11c + CD1c - CD141 + ; CD1c + DC: lineage - HLA-DR + AXL - CD123 - CD11c + CD141 - CD1c + . (B) Comparative analysis of the relative frequencies of DC subsets (AXL + DC, pDCs, CD1c + DC, CD141 + DC, and CD1c - CD141 - DC) in PBMCs between males (n = 46) and females (n = 45). (C) Correlation between donor age and the frequency of pDCs (left) and AXL + DCs (right). Each dot represents an individual donor. (D) The UpSet plot illustrating the integrated comparative analysis of differentially expressed genes (DEGs) in DC subsets between females and males. (E) Functional pathway enrichment analysis of DEGs in CD141 + DC, CD1c + DC, and pDCs, as determined by Ingenuity Pathway Analysis (IPA). The color scale represents the activation z-score, with red indicating pathway activation and blue indicating inhibition. Gray indicates that an activation state could not be determined. (F) Predicted upstream regulators of the DEGs in each DC subset from females compared to males. The color scale represents the activation z-score, with red indicating predicted activation and blue indicating predicted inhibition of the regulator in females. (G) Mean Fluorescence Intensity (MFI) of HLA-DR on CD141 + DC, CD1c + DC, and pDCs, comparing male and female donors. (H) Relative expression of IFN-β in R848-stimulated pDCs from females (n=10) and males (n =10), measured by qPCR, and IFN-beta secretion into the supernatant, quantified by ELISA. Data was shown as mean ± SD. Statistical significance for comparisons between two groups was determined using the Mann-Whitney test. Correlations in (C) were assessed by Spearman’s rank correlation. Significance levels are denoted as follows: ns,not significant; * p < 0.05, ** p < 0.01.

Article Snippet: The total pDCs were isolated from PBMCs using EasySep human pDCs isolation kit (Stemcell, Cat. no17977) and 900 pDCs was plated in 96U plate, cells were stimulated with 10ng/ml TLR7/8 agonist R848 compound (TLRL-R848, MCE) for 24hours.

Techniques: Flow Cytometry, Functional Assay, Activation Assay, Inhibition, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: iScience

Article Title: Comprehensive analysis of neutrophil immunomodulatory properties and FcR dynamics in HIV-1 infection and therapy

doi: 10.1016/j.isci.2026.115431

Figure Lengend Snippet: Activation of human neutrophils stimulated in vitro by Toll-like receptor (TLR) agonists Neutrophils from HD were activated overnight (20h) by TLR4 (LPS) or TLR7/8 (R848) agonists. Activated neutrophils and corresponding cell culture supernatants were collected to evaluate neutrophil viability and phenotypic activation via flow cytometry, as well as to analyze their secretome using bead-based immunoassays. (A) Gating strategy used for all experiments. (B and C) Viability (B) and phenotypic activation (C) by monitoring cell surface markers using flow cytometry. Boxplots (median, first and third quartiles, minimum and maximum) are used to represent selected parameters from 14 independent experiments. (D) Cytokines and chemokines secretion profile. Secretion of soluble cytokines (IL-1β, Il-6, TNF-α, CXCL10, IFN-λ1, IL-8, IL-12p70, IFN-α2, IFN-λ2/3, GM-CSF, IFN-β, IL-10, and IFN-γ) and chemokines (IL-8, CXCL10, CCL11, CCL17, CCL2, CCL5, CCL3, CXCL9, CXCL5, CCL20, CXCL1, CXCL11, and CCL4) was assayed from cell-free cell culture supernatants. Only cytokines/chemokines detected in cell culture supernatants are represented. Values are median with an interquartile range (IQR) of 8 independent measurements. Significance was assigned as follows: ∗ p < 0.05, ∗∗ p < 0,01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 NS: non-stimulated. (B and C) One-way ANOVA with Multiple comparisons (Friedman test). (D) One-way ANOVA with multiple comparisons (Dunn test).

Article Snippet: TLR7/8 agonist: R848 , Invivogen , cat #tlrl-r848.

Techniques: Activation Assay, In Vitro, Cell Culture, Flow Cytometry

Journal: iScience

Article Title: Comprehensive analysis of neutrophil immunomodulatory properties and FcR dynamics in HIV-1 infection and therapy

doi: 10.1016/j.isci.2026.115431

Figure Lengend Snippet: Activation of neutrophils isolated from HD or PLWH stimulated in vitro by TLR agonists LPS (TLR4) and R848 (TLR7/8) or inflammatory cytokines (TNF-α and IFN-γ) Neutrophils from HD were activated overnight (20h) by TLR agonists or pro-inflammatory cytokines. Cell culture supernatants of activated neutrophils were collected to analyze their secretome using bead-based immunoassays, as shown in . (A and B) Cytokines and chemokines secretion profile upon activation by TLR agonists, represented as a heatmap (A) and histograms (B) for each cytokine/chemokine assessed. Values are median with an IQR of at least 9 independent measurements. The heatmap in (A) represents data from all samples shown in (B). (C and D) Cytokine and chemokine secretion profile upon activation by inflammatory cytokines, represented as a heatmap (C) and histograms (D) for each cytokine/chemokine assessed. Values are median with an IQR of at least 9 independent measurements. The heatmap in (C) represents data from all samples shown in (D). Several cytokines and chemokines were only detected in a fraction of samples assayed. Significance was assigned as follows: ∗ p < 0.05 and ∗∗ p < 0.01. Kruskal-Wallis test for unpaired data with additional multiple comparisons, Dunn test.

Article Snippet: TLR7/8 agonist: R848 , Invivogen , cat #tlrl-r848.

Techniques: Activation Assay, Isolation, In Vitro, Cell Culture

Journal: iScience

Article Title: Comprehensive analysis of neutrophil immunomodulatory properties and FcR dynamics in HIV-1 infection and therapy

doi: 10.1016/j.isci.2026.115431

Figure Lengend Snippet: Activation of human neutrophils stimulated in vitro by Toll-like receptor (TLR) agonists Neutrophils from HD were activated overnight (20h) by TLR4 (LPS) or TLR7/8 (R848) agonists. Activated neutrophils and corresponding cell culture supernatants were collected to evaluate neutrophil viability and phenotypic activation via flow cytometry, as well as to analyze their secretome using bead-based immunoassays. (A) Gating strategy used for all experiments. (B and C) Viability (B) and phenotypic activation (C) by monitoring cell surface markers using flow cytometry. Boxplots (median, first and third quartiles, minimum and maximum) are used to represent selected parameters from 14 independent experiments. (D) Cytokines and chemokines secretion profile. Secretion of soluble cytokines (IL-1β, Il-6, TNF-α, CXCL10, IFN-λ1, IL-8, IL-12p70, IFN-α2, IFN-λ2/3, GM-CSF, IFN-β, IL-10, and IFN-γ) and chemokines (IL-8, CXCL10, CCL11, CCL17, CCL2, CCL5, CCL3, CXCL9, CXCL5, CCL20, CXCL1, CXCL11, and CCL4) was assayed from cell-free cell culture supernatants. Only cytokines/chemokines detected in cell culture supernatants are represented. Values are median with an interquartile range (IQR) of 8 independent measurements. Significance was assigned as follows: ∗ p < 0.05, ∗∗ p < 0,01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 NS: non-stimulated. (B and C) One-way ANOVA with Multiple comparisons (Friedman test). (D) One-way ANOVA with multiple comparisons (Dunn test).

Article Snippet: Cells were stimulated for 20h using TLR agonist, inflammatory cytokines or viral determinants under the following conditions: (i) TLR4 agonist (LPS ; Sigma-Aldrich, cat #L4524) or TLR7/8 agonist (R848 ; InvivoGen, cat #tlrl-r848) at 0,5 μg/mL each; (ii) IFN-γ (R&D, cat #285-IF-100) at 50ng/mL or TNF-α (Peprotech, cat #AF-300-01A) at 1ng/mL; (iii) free HIV-1 virions (Ad8 strain, 10 ng/mL of HIV-1 p24) or ICs made with HIV-1 Ad8 and the 10-1074 bNAb (as described below) or the 10-1074 bNAb alone (at 1μg/mL).

Techniques: Activation Assay, In Vitro, Cell Culture, Flow Cytometry

Journal: iScience

Article Title: Comprehensive analysis of neutrophil immunomodulatory properties and FcR dynamics in HIV-1 infection and therapy

doi: 10.1016/j.isci.2026.115431

Figure Lengend Snippet: Activation of neutrophils isolated from HD or PLWH stimulated in vitro by TLR agonists LPS (TLR4) and R848 (TLR7/8) or inflammatory cytokines (TNF-α and IFN-γ) Neutrophils from HD were activated overnight (20h) by TLR agonists or pro-inflammatory cytokines. Cell culture supernatants of activated neutrophils were collected to analyze their secretome using bead-based immunoassays, as shown in . (A and B) Cytokines and chemokines secretion profile upon activation by TLR agonists, represented as a heatmap (A) and histograms (B) for each cytokine/chemokine assessed. Values are median with an IQR of at least 9 independent measurements. The heatmap in (A) represents data from all samples shown in (B). (C and D) Cytokine and chemokine secretion profile upon activation by inflammatory cytokines, represented as a heatmap (C) and histograms (D) for each cytokine/chemokine assessed. Values are median with an IQR of at least 9 independent measurements. The heatmap in (C) represents data from all samples shown in (D). Several cytokines and chemokines were only detected in a fraction of samples assayed. Significance was assigned as follows: ∗ p < 0.05 and ∗∗ p < 0.01. Kruskal-Wallis test for unpaired data with additional multiple comparisons, Dunn test.

Article Snippet: Cells were stimulated for 20h using TLR agonist, inflammatory cytokines or viral determinants under the following conditions: (i) TLR4 agonist (LPS ; Sigma-Aldrich, cat #L4524) or TLR7/8 agonist (R848 ; InvivoGen, cat #tlrl-r848) at 0,5 μg/mL each; (ii) IFN-γ (R&D, cat #285-IF-100) at 50ng/mL or TNF-α (Peprotech, cat #AF-300-01A) at 1ng/mL; (iii) free HIV-1 virions (Ad8 strain, 10 ng/mL of HIV-1 p24) or ICs made with HIV-1 Ad8 and the 10-1074 bNAb (as described below) or the 10-1074 bNAb alone (at 1μg/mL).

Techniques: Activation Assay, Isolation, In Vitro, Cell Culture