Journal: PLoS ONE

Article Title: Lipoglycans Contribute to Innate Immune Detection of Mycobacteria

doi: 10.1371/journal.pone.0028476

Figure Lengend Snippet: A, B, E, F) NF-κB activation in HEK (A, E) and THP-1 (B, F) cells. C, D, G) IL-8 production by THP-1 (C, G) and human monocyte-derived dendritic (D) cells. Cells were incubated with bacteria at MOI of 1. In F and G, cells were pre-incubated for 30 min at 37°C before bacteria addition with various antibodies, anti-TLR2 and isotype controls, at a concentration of 5 µg/ml. The results are mean ± SD of triplicate wells and are representative of at least three separate experiments using independent bacterial cultures and different MOI. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. In A, B, C and D), P value is given vs pMV. n.i., not induced.

Article Snippet: THP-1-Blue TM cells (Invivogen), derivatives of THP-1 monocyte/macrophage human cells that stably express a NF-κB-inducible reporter system (secreted alkaline phosphatase), were added at 10 5 cells per well in 96-wells plates in HEK-Blue TM Detection medium (for NF-κB activation assay) or differentiated with 20 ng/ml of PMA for 24 h in RPMI 1640 culture medium (Lonza, Verviers, Belgium) (for IL-8 production assay).

Techniques: Activation Assay, Derivative Assay, Incubation, Bacteria, Concentration Assay

Journal: PLoS ONE

Article Title: Lipoglycans Contribute to Innate Immune Detection of Mycobacteria

doi: 10.1371/journal.pone.0028476

Figure Lengend Snippet: A, B, E, F) NF-κB activation in HEK-TLR2 (A, E) and THP-1 (B, F) cells. C, D, G, H) IL-8 production by THP-1 (C, G) and human monocyte-derived dendritic (D, H) cells. Cells were incubated with bacteria at MOI of 1. The results are mean ± SD of triplicate wells and are representative of at least three separate experiments using independent bacterial cultures and different MOI. *, P<0.05; **, P<0.01; ***, P<0.001. n.i., not induced.

Article Snippet: THP-1-Blue TM cells (Invivogen), derivatives of THP-1 monocyte/macrophage human cells that stably express a NF-κB-inducible reporter system (secreted alkaline phosphatase), were added at 10 5 cells per well in 96-wells plates in HEK-Blue TM Detection medium (for NF-κB activation assay) or differentiated with 20 ng/ml of PMA for 24 h in RPMI 1640 culture medium (Lonza, Verviers, Belgium) (for IL-8 production assay).

Techniques: Activation Assay, Derivative Assay, Incubation, Bacteria

Journal: PLoS ONE

Article Title: Lipoteichoic Acid in Streptomyces hygroscopicus : Structural Model and Immunomodulatory Activities

doi: 10.1371/journal.pone.0026316

Figure Lengend Snippet: A. THP-1 cells were stimulated with 1 or 10 µg.ml −1 of ShLTA, SaLTA or Pam 3 CSK 4 . NF-κB activation was determined by reading OD at 630 nm. B, C. THP-1 cells were differentiated with 20 ng.ml −1 of PMA for 24 h and then stimulated with 1 or 10 µg.ml −1 of ShLTA, SaLTA or Pam 3 CSK 4 . IL-6 and TNF-α were assayed in the supernatant by sandwich ELISA. The results are mean ± SD and are representative of three separate experiments.

Article Snippet: THP-1-Blue cell line (InvivoGen), a derivative of THP-1 monocyte/macrophage human cells that stably expresses a NF-κB-inducible reporter system (secreted alkaline phosphatase) was used according to the manufacturer's instructions.

Techniques: Activation Assay, Sandwich ELISA

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: THP-1-derived M1 and M2 MΦs were generated by differentiating THP-1 monocytes with either 25 ng/ml phorbol 12-myristate 13-acetate (PMA) for 3 days or 10 nM 1,25-(OH) 2 vitamin D 3 for 7 days, respectively. M1 (bold) and M2 (shaded) MΦ subsets were stimulated with either 100 ng/ml PG-LPS (a, b and c) or 1×10 7 cells/ml HKPG (d, e and f). Cytokine production is expressed as the mean ± SD in pg/ml for TNFα (a & d), IL-1β (b & e) and IL-6 (c & f). Data displayed represents triplicate samples for n = 3 replicate experiments. Significant differences in cytokine production between activated M1 and M2 MΦs are indicated as *p<0.05, **p<0.01, ***P<0.001 and ns, not significant.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques: Derivative Assay, Generated

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: THP-1-derived CD14-high- and CD14-low-expressing (CD14 hi and CD14 lo ) M1 and M2 MΦs were generated by differentiating CD14 + and CD14 − stable transfectant THP-1-blue monocytes with either 25 ng/ml phorbol 12-myristate 13-acetate (PMA) for 3 days or 10 nM 1,25-(OH) 2 vitamin D 3 for 7 days, respectively. CD14 hi /CD14 lo M1 (bold) and M2 (shaded) MΦ subsets were stimulated with either 100ng/ml PG-LPS (a, b, c & d) or 1×10 7 cells/ml HKPG (e, f, g & h). Cytokine production is expressed as the mean ± SD in pg/ml for TNFα (a & e), IL-1β (b & f), IL-6 (c & g) and IL-10 (d & h). Data displayed represents triplicate samples for n = 3 replicate experiments. Significant differences in cytokine production between activated CD14 hi and CD14 lo MΦs are indicated as *p<0.05, **p<0.01, ***P<0.001 and ns, not significant.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques: Derivative Assay, Expressing, Generated, Transfection

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: PG-LPS exhibits weak endotoxin activity in CD14 hi/lo M1/M2 macrophages.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: CD14 lo (bold) and CD14 hi (shaded) M1 and M2 MΦ subsets were stimulated with either 100 ng/ml PG-LPS or 1×10 7 cells/ml HKPG. NFκB activation is expressed as the mean absorbance units A 620nm ± SD for M1 (a) and M2 (b) MΦ subsets. Data displayed represents triplicate samples for n = 3 replicate experiments. Significant differences in NFκB activation between CD14 hi and CD14 lo MΦs are indicated as *p<0.05, **p<0.01 and ns, not significant.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: CD14 lo M1 (a) CD14 hi M1 (b), CD14 lo M2 (c) and CD14 hi M2 (d) MΦ subsets were pre-stimulated (tolerised) with either 100 ng/ml PG-LPS (unshaded) or 1×10 7 cells/ml HKPG (shaded) for 24 hours prior to stimulation with PG-LPS or HKPG and incubated for a further 18 hours (untolerised controls indicated in bold). NFκB activation is expressed as the mean absorbance units A 620nm ± SD for the CD14 hi/lo M1 and M2 MΦ subsets. Data displayed represents triplicate samples for n = 3 replicate experiments. Significant effects compared to the un-tolerised stimulus control (bold) for each MΦ subset are indicated as *p<0.05, ***p<0.001 and ns, not significant.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques: Incubation, Activation Assay

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: CD14 lo M1 (a) CD14 hi M1 (b), CD14 lo M2 (c) and CD14 hi M2 (d) MΦ subsets were pre-stimulated (tolerised) with either 100 ng/ml PG-LPS (unshaded) or 1×10 7 cells/ml HKPG (shaded) for 24 hours prior to stimulation with PG-LPS or HKPG and incubated for a further 18 hours (untolerised controls indicated in bold). Pro-inflammatory TNFα cytokine production is expressed in pg/ml as the mean ± SD for the CD14 hi/lo M1 and M2 MΦ subsets. Data displayed represents triplicate samples for n = 3 replicate experiments. Significant effects compared to the un-tolerised stimulus control (bold) for each MΦ subset are indicated as ***p<0.001 and ns, not significant.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: CD14 lo M1 (a) CD14 hi M1 (b), CD14 lo M2 (c) and CD14 hi M2 (d) MΦ subsets were pre-stimulated (tolerised) with either 100 ng/ml PG-LPS (unshaded) or 1×10 7 cells/ml HKPG (shaded) for 24 hours prior to stimulation with PG-LPS or HKPG and incubated for a further 18 hours (untolerised controls indicated in bold). Anti-inflammatory IL-10 cytokine production is expressed in pg/ml as the mean ± SD for the CD14 hi/lo M1 and M2 MΦ subsets. Data displayed represents triplicate samples for n = 3 replicate experiments. Significant effects compared to the un-tolerised stimulus controls (bold) for each MΦ subset are are indicated as*p<0.05, **p<0.01, ***p<0.001 and ns, not significant.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: CD14 hi/lo M1 and M2 MΦ cytokines are differentially tolerised by P.gingivalis and LTA PAMPs.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques:

Journal: PLoS ONE

Article Title: Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

doi: 10.1371/journal.pone.0067955

Figure Lengend Snippet: Peptidoglycan differentially cross-tolerises P. gingivalis -stimulated CD14 hi/lo M1 and M2 MΦ subsets.

Article Snippet: The THP-1 NFκB reporter cell lines THP-1Blue (CD14 lo ) and THP-1Blue-CD14 (CD14 hi ) (Autogen Bioclear, Calne, UK) were maintained in R10 medium in the presence of the selection antibiotics, zeocin (200 µg/ml) only (CD14 lo ) or 200 µg/ml zeocin and 10 µg/ml blastocidin (CD14 hi ) (Autogen Bioclear, Calne, UK).

Techniques: