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  • 95
    InvivoGen hek blue htlr9
    CpG 2395, DNA 2395, and TNA 2395 activate Ramos and Raji cells, while only CpG 2395 induces SEAP reporter activity in HEK-Blue <t>hTLR9</t> cells. (A, B) Ramos and Raji cells were plated at a density of 2 × 105 cells in 12-well plates and stimulated with the indicated ligands at the indicated concentrations. After 72 h, cells were harvested for flow cytometric analysis of surface-expressed CD86 expressing using a PE-labeled antibody specific for CD86. A PE-labeled isotype antibody was included as a control. Data are shown relative to the untreated control for Ramos (A) and Raji (B) cells. Error bars represent the standard deviation between two replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. (C, D) HEK-Blue hTLR9 cells were plated at a density of 5 × 104 in 96-well plates and allowed to adhere overnight. Cells were then stimulated with the indicated ligands at the indicated concentrations. After 24 (A) or 72 (B) hours, supernatants were harvested and tested for SEAP activity using QUANTI-Blue reagent. Samples were assayed in triplicate. Error bars represent standard deviations for three replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. PE, phycoerythrin; SEAP, secreted alkaline phosphatase.
    Hek Blue Htlr9, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    R&D Systems antibodies against human tlr9
    CpG 2395, DNA 2395, and TNA 2395 activate Ramos and Raji cells, while only CpG 2395 induces SEAP reporter activity in HEK-Blue <t>hTLR9</t> cells. (A, B) Ramos and Raji cells were plated at a density of 2 × 105 cells in 12-well plates and stimulated with the indicated ligands at the indicated concentrations. After 72 h, cells were harvested for flow cytometric analysis of surface-expressed CD86 expressing using a PE-labeled antibody specific for CD86. A PE-labeled isotype antibody was included as a control. Data are shown relative to the untreated control for Ramos (A) and Raji (B) cells. Error bars represent the standard deviation between two replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. (C, D) HEK-Blue hTLR9 cells were plated at a density of 5 × 104 in 96-well plates and allowed to adhere overnight. Cells were then stimulated with the indicated ligands at the indicated concentrations. After 24 (A) or 72 (B) hours, supernatants were harvested and tested for SEAP activity using QUANTI-Blue reagent. Samples were assayed in triplicate. Error bars represent standard deviations for three replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. PE, phycoerythrin; SEAP, secreted alkaline phosphatase.
    Antibodies Against Human Tlr9, supplied by R&D Systems, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    InvivoGen htlr9 cells
    Culture supernatants from macrolide-treated MRSP cells induce less TLR2 and NOD2 activation. MRSP strain NU4471 was grown in the presence or absence of 5 μg/mL azithromycin (AZM) or erythromycin (ERY) until it reached the stationary phase. (A to E) HEK-Blue hTLR-2 cells (A), HEK-Blue hNOD2 cells (B), HEK-Blue hTLR4 cells (C), HEK-Blue <t>hTLR9</t> cells (D), and HEK-Blue null2 negative-control cells (E) were incubated with the culture supernatant (Sup) from untreated or macrolide-treated MRSP cells for 12 to 20 h (final concentrations of macrolides in macrolide-treated groups, 0.5 μg/mL). (F to J) HEK-Blue hTLR-2 cells (F), HEK-Blue hNOD2 cells (G), HEK-Blue hTLR4 cells (H), HEK-Blue hTLR9 cells (I), and HEK-Blue null2 negative-control cells (J) were exposed to supernatant from untreated MRSP cells in the presence or absence of 0.5 μg/mL AZM or ERY for 12 h. As controls, cells were also exposed to RPMI 1640 medium containing 0.5 μg/mL AZM or ERY. (A to J) Secreted alkaline phosphatase (SEAP) levels were quantified using spectrophotometry at 620 nm. Data represent the mean values ± SD from quintuplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparison test. †, significant difference compared with the control at P < 0.05; *, significant difference between the indicated groups at P < 0.05. Ctrl, control; LPS, lipopolysaccharide; MDP, muramyl dipeptide; MRSP, macrolide-resistant S. pneumoniae ; ODN, CpG ODN; Pam, Pam3CSK4.
    Htlr9 Cells, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    InvivoGen human tlr9 agonist kit
    WT and <t>Tlr9–/–</t> mice were subjected to CLP. PLF was collected at 18 hours after CLP. (A) Percentages of indicated immune cells were measured by flow cytometry. (B) TLR9 expression in indicated peritoneal cells. MFI for TLR9 expression was measured by flow cytometry. (C–G) Peritoneal cell counts of (C) B cells, (D) macrophages, (E) DCs, (F) neutrophils, and (G) T cells in control and after CLP. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t tests.
    Human Tlr9 Agonist Kit, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    InvivoGen hek-blue-lucia htlr9
    WT and <t>Tlr9–/–</t> mice were subjected to CLP. PLF was collected at 18 hours after CLP. (A) Percentages of indicated immune cells were measured by flow cytometry. (B) TLR9 expression in indicated peritoneal cells. MFI for TLR9 expression was measured by flow cytometry. (C–G) Peritoneal cell counts of (C) B cells, (D) macrophages, (E) DCs, (F) neutrophils, and (G) T cells in control and after CLP. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t tests.
    Hek Blue Lucia Htlr9, supplied by InvivoGen, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hek-blue-lucia htlr9/product/InvivoGen
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    Image Search Results


    CpG 2395, DNA 2395, and TNA 2395 activate Ramos and Raji cells, while only CpG 2395 induces SEAP reporter activity in HEK-Blue hTLR9 cells. (A, B) Ramos and Raji cells were plated at a density of 2 × 105 cells in 12-well plates and stimulated with the indicated ligands at the indicated concentrations. After 72 h, cells were harvested for flow cytometric analysis of surface-expressed CD86 expressing using a PE-labeled antibody specific for CD86. A PE-labeled isotype antibody was included as a control. Data are shown relative to the untreated control for Ramos (A) and Raji (B) cells. Error bars represent the standard deviation between two replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. (C, D) HEK-Blue hTLR9 cells were plated at a density of 5 × 104 in 96-well plates and allowed to adhere overnight. Cells were then stimulated with the indicated ligands at the indicated concentrations. After 24 (A) or 72 (B) hours, supernatants were harvested and tested for SEAP activity using QUANTI-Blue reagent. Samples were assayed in triplicate. Error bars represent standard deviations for three replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. PE, phycoerythrin; SEAP, secreted alkaline phosphatase.

    Journal: Nucleic Acid Therapeutics

    Article Title: Activation of Innate Immune Responses by a CpG Oligonucleotide Sequence Composed Entirely of Threose Nucleic Acid

    doi: 10.1089/nat.2018.0751

    Figure Lengend Snippet: CpG 2395, DNA 2395, and TNA 2395 activate Ramos and Raji cells, while only CpG 2395 induces SEAP reporter activity in HEK-Blue hTLR9 cells. (A, B) Ramos and Raji cells were plated at a density of 2 × 105 cells in 12-well plates and stimulated with the indicated ligands at the indicated concentrations. After 72 h, cells were harvested for flow cytometric analysis of surface-expressed CD86 expressing using a PE-labeled antibody specific for CD86. A PE-labeled isotype antibody was included as a control. Data are shown relative to the untreated control for Ramos (A) and Raji (B) cells. Error bars represent the standard deviation between two replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. (C, D) HEK-Blue hTLR9 cells were plated at a density of 5 × 104 in 96-well plates and allowed to adhere overnight. Cells were then stimulated with the indicated ligands at the indicated concentrations. After 24 (A) or 72 (B) hours, supernatants were harvested and tested for SEAP activity using QUANTI-Blue reagent. Samples were assayed in triplicate. Error bars represent standard deviations for three replicates within a single experiment. A representative experiment is shown in each panel. Experiments were performed 3 times. PE, phycoerythrin; SEAP, secreted alkaline phosphatase.

    Article Snippet: The human cell line expressing TLR9, HEK-Blue hTLR9 (Invivogen), was maintained according to the manufacturer's instructions.

    Techniques: Activity Assay, Expressing, Labeling, Standard Deviation

    Culture supernatants from macrolide-treated MRSP cells induce less TLR2 and NOD2 activation. MRSP strain NU4471 was grown in the presence or absence of 5 μg/mL azithromycin (AZM) or erythromycin (ERY) until it reached the stationary phase. (A to E) HEK-Blue hTLR-2 cells (A), HEK-Blue hNOD2 cells (B), HEK-Blue hTLR4 cells (C), HEK-Blue hTLR9 cells (D), and HEK-Blue null2 negative-control cells (E) were incubated with the culture supernatant (Sup) from untreated or macrolide-treated MRSP cells for 12 to 20 h (final concentrations of macrolides in macrolide-treated groups, 0.5 μg/mL). (F to J) HEK-Blue hTLR-2 cells (F), HEK-Blue hNOD2 cells (G), HEK-Blue hTLR4 cells (H), HEK-Blue hTLR9 cells (I), and HEK-Blue null2 negative-control cells (J) were exposed to supernatant from untreated MRSP cells in the presence or absence of 0.5 μg/mL AZM or ERY for 12 h. As controls, cells were also exposed to RPMI 1640 medium containing 0.5 μg/mL AZM or ERY. (A to J) Secreted alkaline phosphatase (SEAP) levels were quantified using spectrophotometry at 620 nm. Data represent the mean values ± SD from quintuplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparison test. †, significant difference compared with the control at P < 0.05; *, significant difference between the indicated groups at P < 0.05. Ctrl, control; LPS, lipopolysaccharide; MDP, muramyl dipeptide; MRSP, macrolide-resistant S. pneumoniae ; ODN, CpG ODN; Pam, Pam3CSK4.

    Journal: Microbiology Spectrum

    Article Title: Macrolides Decrease the Proinflammatory Activity of Macrolide-Resistant Streptococcus pneumoniae

    doi: 10.1128/spectrum.00148-23

    Figure Lengend Snippet: Culture supernatants from macrolide-treated MRSP cells induce less TLR2 and NOD2 activation. MRSP strain NU4471 was grown in the presence or absence of 5 μg/mL azithromycin (AZM) or erythromycin (ERY) until it reached the stationary phase. (A to E) HEK-Blue hTLR-2 cells (A), HEK-Blue hNOD2 cells (B), HEK-Blue hTLR4 cells (C), HEK-Blue hTLR9 cells (D), and HEK-Blue null2 negative-control cells (E) were incubated with the culture supernatant (Sup) from untreated or macrolide-treated MRSP cells for 12 to 20 h (final concentrations of macrolides in macrolide-treated groups, 0.5 μg/mL). (F to J) HEK-Blue hTLR-2 cells (F), HEK-Blue hNOD2 cells (G), HEK-Blue hTLR4 cells (H), HEK-Blue hTLR9 cells (I), and HEK-Blue null2 negative-control cells (J) were exposed to supernatant from untreated MRSP cells in the presence or absence of 0.5 μg/mL AZM or ERY for 12 h. As controls, cells were also exposed to RPMI 1640 medium containing 0.5 μg/mL AZM or ERY. (A to J) Secreted alkaline phosphatase (SEAP) levels were quantified using spectrophotometry at 620 nm. Data represent the mean values ± SD from quintuplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparison test. †, significant difference compared with the control at P < 0.05; *, significant difference between the indicated groups at P < 0.05. Ctrl, control; LPS, lipopolysaccharide; MDP, muramyl dipeptide; MRSP, macrolide-resistant S. pneumoniae ; ODN, CpG ODN; Pam, Pam3CSK4.

    Article Snippet: The selection antibiotics used were 100 μg/mL Zeocin (InvivoGen) for HEK-Blue null2 cells, HEK-Blue selection (InvivoGen) for hTLR2 and hTLR4 cells, 10 μg/mL blasticidin (InvivoGen) and 100 μg/mL Zeocin for hTLR9 cells, and 30 μg/mL blasticidin and 100 μg/mL Zeocin for hNOD2 cells.

    Techniques: Activation Assay, Negative Control, Incubation, Spectrophotometry, Comparison

    Treatment of bacterial cells with macrolides induces less TLR2 activation. MRSP strain NU4471 was grown in the presence or absence of 5 μg/mL azithromycin (AZM) or erythromycin (ERY) until it reached the stationary phase. Pneumococcal cells were killed by treatment with 70% ethanol. (A) HEK-Blue hTLR-2 cells were stimulated with ethanol-killed S. pneumoniae (Spn) for 12 h. (B) HEK-Blue hNOD2 cells were stimulated with Spn for 18 h. (C) HEK-Blue hTLR4 cells were stimulated with Spn for 20 h. (D) HEK-Blue hTLR9 cells were stimulated with Spn for 20 h. (E) HEK-Blue null2 negative-control cells were stimulated with Spn for 17 h. (A to E) Secreted alkaline phosphatase (SEAP) levels were quantified using spectrophotometry at 620 nm. Data represent the mean values ± SD from quintuplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparison test. †, significant difference compared with the control at P < 0.05; *, significant difference between the indicated groups at P < 0.05. Ctrl, control; LPS, lipopolysaccharide; MRSP, macrolide-resistant S. pneumoniae ; MDP, muramyl dipeptide; ODN, CpG ODN; Pam, Pam3CSK4.

    Journal: Microbiology Spectrum

    Article Title: Macrolides Decrease the Proinflammatory Activity of Macrolide-Resistant Streptococcus pneumoniae

    doi: 10.1128/spectrum.00148-23

    Figure Lengend Snippet: Treatment of bacterial cells with macrolides induces less TLR2 activation. MRSP strain NU4471 was grown in the presence or absence of 5 μg/mL azithromycin (AZM) or erythromycin (ERY) until it reached the stationary phase. Pneumococcal cells were killed by treatment with 70% ethanol. (A) HEK-Blue hTLR-2 cells were stimulated with ethanol-killed S. pneumoniae (Spn) for 12 h. (B) HEK-Blue hNOD2 cells were stimulated with Spn for 18 h. (C) HEK-Blue hTLR4 cells were stimulated with Spn for 20 h. (D) HEK-Blue hTLR9 cells were stimulated with Spn for 20 h. (E) HEK-Blue null2 negative-control cells were stimulated with Spn for 17 h. (A to E) Secreted alkaline phosphatase (SEAP) levels were quantified using spectrophotometry at 620 nm. Data represent the mean values ± SD from quintuplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparison test. †, significant difference compared with the control at P < 0.05; *, significant difference between the indicated groups at P < 0.05. Ctrl, control; LPS, lipopolysaccharide; MRSP, macrolide-resistant S. pneumoniae ; MDP, muramyl dipeptide; ODN, CpG ODN; Pam, Pam3CSK4.

    Article Snippet: The selection antibiotics used were 100 μg/mL Zeocin (InvivoGen) for HEK-Blue null2 cells, HEK-Blue selection (InvivoGen) for hTLR2 and hTLR4 cells, 10 μg/mL blasticidin (InvivoGen) and 100 μg/mL Zeocin for hTLR9 cells, and 30 μg/mL blasticidin and 100 μg/mL Zeocin for hNOD2 cells.

    Techniques: Activation Assay, Negative Control, Spectrophotometry, Comparison

    WT and Tlr9–/– mice were subjected to CLP. PLF was collected at 18 hours after CLP. (A) Percentages of indicated immune cells were measured by flow cytometry. (B) TLR9 expression in indicated peritoneal cells. MFI for TLR9 expression was measured by flow cytometry. (C–G) Peritoneal cell counts of (C) B cells, (D) macrophages, (E) DCs, (F) neutrophils, and (G) T cells in control and after CLP. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t tests.

    Journal: The Journal of Clinical Investigation

    Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

    doi: 10.1172/JCI127542

    Figure Lengend Snippet: WT and Tlr9–/– mice were subjected to CLP. PLF was collected at 18 hours after CLP. (A) Percentages of indicated immune cells were measured by flow cytometry. (B) TLR9 expression in indicated peritoneal cells. MFI for TLR9 expression was measured by flow cytometry. (C–G) Peritoneal cell counts of (C) B cells, (D) macrophages, (E) DCs, (F) neutrophils, and (G) T cells in control and after CLP. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t tests.

    Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

    Techniques: Flow Cytometry, Expressing

    (A–D) WT and Tlr9–/– mice were subjected to CLP. Plasma and PLF were collected at 18 hours after CLP. (A) Peritoneal B-1 cell numbers. (B) Plasma IgM levels. (C) Peritoneal IgM levels. (D) Peritoneal GM-CSF levels. (E–G) Tlr9–/– mice were treated with CD19 neutralizing antibodies (10 mg/mouse) or control IgG (10 mg/mouse) at 24 hours before CLP. PLF was collected at 18 hours after CLP. (E) Seven-day survival after CLP. Data are from 2 separate experiments. n = 10. *P < 0.05, log-rank test. (F) Bacterial clearance in PLF. Data are from 2 separate experiments. Symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. (G) Peritoneal IgM levels. For A–D and G, data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t test.

    Journal: The Journal of Clinical Investigation

    Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

    doi: 10.1172/JCI127542

    Figure Lengend Snippet: (A–D) WT and Tlr9–/– mice were subjected to CLP. Plasma and PLF were collected at 18 hours after CLP. (A) Peritoneal B-1 cell numbers. (B) Plasma IgM levels. (C) Peritoneal IgM levels. (D) Peritoneal GM-CSF levels. (E–G) Tlr9–/– mice were treated with CD19 neutralizing antibodies (10 mg/mouse) or control IgG (10 mg/mouse) at 24 hours before CLP. PLF was collected at 18 hours after CLP. (E) Seven-day survival after CLP. Data are from 2 separate experiments. n = 10. *P < 0.05, log-rank test. (F) Bacterial clearance in PLF. Data are from 2 separate experiments. Symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. (G) Peritoneal IgM levels. For A–D and G, data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired, 2-tailed Student’s t test.

    Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

    Techniques: MANN-WHITNEY

    (A) WT and Tlr9–/– mice were subjected to CLP. PLF was collected at 18 hours after CLP. Peritoneal CXCL13 levels were assessed using ELISA. (B–F) WT and Tlr9–/– mice were treated with CXCL13 neutralizing antibodies (10 mg/mouse) or control IgG (10 mg/mouse) immediately after CLP. PLF and plasma were collected at 18 hours after CLP. (B) Peritoneal B-1 cell numbers. (C) Peritoneal IgM levels. (D) Bacterial load in PLF. (E) Plasma IL-6 levels. (F) Seven-day survival. For A–C and E, data are shown as mean ± SD. Symbols represent individual mice. *P < 0.05; **P < 0.01, unpaired, 2-tailed Student’s t tests. For D, symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. For F, n = 13–19/group as indicated. *P < 0.05 versus Tlr9–/– IgG, log-rank test. (G–K) WT mice were treated with recombinant CXCL13 (10 mg/mouse) or PBS immediately after CLP. (G) Peritoneal B-1 cell number. (H) Peritoneal IgM levels. (I) Bacterial load in PLF. (J) Plasma IL-6 levels. (K) Seven-day survival. For G, H, and J, data are shown as mean ± SD. Symbols represent individual mice. *P < 0.05, unpaired, 2-tailed Student’s t test. For I, symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. For K, n = 10/group. *P < 0.05, log-rank test.

    Journal: The Journal of Clinical Investigation

    Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

    doi: 10.1172/JCI127542

    Figure Lengend Snippet: (A) WT and Tlr9–/– mice were subjected to CLP. PLF was collected at 18 hours after CLP. Peritoneal CXCL13 levels were assessed using ELISA. (B–F) WT and Tlr9–/– mice were treated with CXCL13 neutralizing antibodies (10 mg/mouse) or control IgG (10 mg/mouse) immediately after CLP. PLF and plasma were collected at 18 hours after CLP. (B) Peritoneal B-1 cell numbers. (C) Peritoneal IgM levels. (D) Bacterial load in PLF. (E) Plasma IL-6 levels. (F) Seven-day survival. For A–C and E, data are shown as mean ± SD. Symbols represent individual mice. *P < 0.05; **P < 0.01, unpaired, 2-tailed Student’s t tests. For D, symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. For F, n = 13–19/group as indicated. *P < 0.05 versus Tlr9–/– IgG, log-rank test. (G–K) WT mice were treated with recombinant CXCL13 (10 mg/mouse) or PBS immediately after CLP. (G) Peritoneal B-1 cell number. (H) Peritoneal IgM levels. (I) Bacterial load in PLF. (J) Plasma IL-6 levels. (K) Seven-day survival. For G, H, and J, data are shown as mean ± SD. Symbols represent individual mice. *P < 0.05, unpaired, 2-tailed Student’s t test. For I, symbols represent individual mice. *P < 0.05, nonparametric Mann-Whitney U test. For K, n = 10/group. *P < 0.05, log-rank test.

    Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

    Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Recombinant

    (A–C) WT and Tlr9–/– mice were subjected to CLP. PLF and mesenteric adipose tissue were collected at18 hours after CLP. (A and B) Cxcl13 expression in peritoneal cells (A) and mesenteric adipose tissue (B) were assessed using quantitative PCR. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05, 2-tailed Student’s t test. (C) Immunofluorescence staining of CXCL13 (red), CD45 (green), CD11c (white), and nucleus (blue) in mesenteric adipose tissue. Scale bar: 5 μm. (D and E) Cxcl13 expression in mouse FRCs. WT and Tlr9–/– FRCs were isolated from mesenteric adipose tissues and expanded ex vivo. FRCs were stimulated with indicated TLR ligands (ODN1585, 5 μM; ODN1826, 5 μM; ODN2395, 5 μM, LPS, 1 μg/mL; PIC, 20 μg/mL) for 18 hours. Cxcl13 expression was assessed using quantitative PCR. Data are shown as mean ± SD from 1 representative experiment. Experiments were performed 3 times. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post hoc analysis.

    Journal: The Journal of Clinical Investigation

    Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

    doi: 10.1172/JCI127542

    Figure Lengend Snippet: (A–C) WT and Tlr9–/– mice were subjected to CLP. PLF and mesenteric adipose tissue were collected at18 hours after CLP. (A and B) Cxcl13 expression in peritoneal cells (A) and mesenteric adipose tissue (B) were assessed using quantitative PCR. Data are shown as mean ± SD from 2 separate experiments. Symbols represent individual mice. *P < 0.05, 2-tailed Student’s t test. (C) Immunofluorescence staining of CXCL13 (red), CD45 (green), CD11c (white), and nucleus (blue) in mesenteric adipose tissue. Scale bar: 5 μm. (D and E) Cxcl13 expression in mouse FRCs. WT and Tlr9–/– FRCs were isolated from mesenteric adipose tissues and expanded ex vivo. FRCs were stimulated with indicated TLR ligands (ODN1585, 5 μM; ODN1826, 5 μM; ODN2395, 5 μM, LPS, 1 μg/mL; PIC, 20 μg/mL) for 18 hours. Cxcl13 expression was assessed using quantitative PCR. Data are shown as mean ± SD from 1 representative experiment. Experiments were performed 3 times. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post hoc analysis.

    Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Isolation, Ex Vivo

    (A) Schematic timeline of experimental set and analysis for adoptive transfer studies. (B and C) Mice were subjected to CLP. WT or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. at 1 hour after CLP. (B) Bacterial load in PLF at 18 hours after CLP. (C) Circulating IL-6 levels at 18 hours after CLP. Data are shown as mean ± SD from 2 separate experiments. *P < 0.05, unpaired, 2-tailed Student’s t test. (D and E) Seven-day survival. (D) Mice were subjected to CLP. PBS, WT, or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. 1 hour after CLP. n = 13 in PBS group; n = 19 in WT FRC group; n = 22 in Tlr9–/– FRC group. (E) Mice were subjected to CLP. PBS, WT, or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. at 12 hours after CLP. n = 10 in PBS group; n = 14 in WT FRC group; n = 15 in Tlr9–/– FRC group. *P < 0.05 vs. PBS, log-rank test. Data are from 3 separate experiments for D and 2 separate experiments for E. Statistical differences were determined using the log-rank test.

    Journal: The Journal of Clinical Investigation

    Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

    doi: 10.1172/JCI127542

    Figure Lengend Snippet: (A) Schematic timeline of experimental set and analysis for adoptive transfer studies. (B and C) Mice were subjected to CLP. WT or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. at 1 hour after CLP. (B) Bacterial load in PLF at 18 hours after CLP. (C) Circulating IL-6 levels at 18 hours after CLP. Data are shown as mean ± SD from 2 separate experiments. *P < 0.05, unpaired, 2-tailed Student’s t test. (D and E) Seven-day survival. (D) Mice were subjected to CLP. PBS, WT, or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. 1 hour after CLP. n = 13 in PBS group; n = 19 in WT FRC group; n = 22 in Tlr9–/– FRC group. (E) Mice were subjected to CLP. PBS, WT, or Tlr9–/– FRCs (2 × 105/ mouse) were injected i.p. at 12 hours after CLP. n = 10 in PBS group; n = 14 in WT FRC group; n = 15 in Tlr9–/– FRC group. *P < 0.05 vs. PBS, log-rank test. Data are from 3 separate experiments for D and 2 separate experiments for E. Statistical differences were determined using the log-rank test.

    Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

    Techniques: Adoptive Transfer Assay, Injection

    Human FRCs were isolated from adipose tissue and expanded ex vivo. (A) TLR9 expression in human FRCs. TLR9 expression was assessed using flow cytometry. Numbers indicate percentage of FRCs (CD45–CD31–PDPN+). (B) Chemokine expression in human FRCs. FRCs were stimulated with indicated TLR ligands (ODN2216, 5 μM; LPS, 1 μg/mL; PIC, 20 μg/mL) for 18 hours. Indicated chemokine expression was assessed using quantitative PCR. Data are shown as mean ± SD from 1 representative experiment. Experiments were performed 3 times. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post hoc analysis.

    Journal: The Journal of Clinical Investigation

    Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

    doi: 10.1172/JCI127542

    Figure Lengend Snippet: Human FRCs were isolated from adipose tissue and expanded ex vivo. (A) TLR9 expression in human FRCs. TLR9 expression was assessed using flow cytometry. Numbers indicate percentage of FRCs (CD45–CD31–PDPN+). (B) Chemokine expression in human FRCs. FRCs were stimulated with indicated TLR ligands (ODN2216, 5 μM; LPS, 1 μg/mL; PIC, 20 μg/mL) for 18 hours. Indicated chemokine expression was assessed using quantitative PCR. Data are shown as mean ± SD from 1 representative experiment. Experiments were performed 3 times. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Bonferroni’s post hoc analysis.

    Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

    Techniques: Isolation, Ex Vivo, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

    Activation of TLR9 signaling in FRCs suppresses the production of chemokines, which are essential for recruiting B cells, T cells, macrophages, and neutrophils into the peritoneal cavity as well as driving the formation of FALCs.

    Journal: The Journal of Clinical Investigation

    Article Title: TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity

    doi: 10.1172/JCI127542

    Figure Lengend Snippet: Activation of TLR9 signaling in FRCs suppresses the production of chemokines, which are essential for recruiting B cells, T cells, macrophages, and neutrophils into the peritoneal cavity as well as driving the formation of FALCs.

    Article Snippet: LPS-EK (LPS from E . coli K12), PIC low molecular weight (LMW), Mouse TLR9 Agonist Kit, and Human TLR9 Agonist Kit were purchased from InvivoGen.

    Techniques: Activation Assay