Journal: PLoS ONE

Article Title: Production of Factor VIII by Human Liver Sinusoidal Endothelial Cells Transplanted in Immunodeficient uPA Mice

doi: 10.1371/journal.pone.0077255

Figure Lengend Snippet: VEGF levels in the liver are shown as ratio of the amount of VEGF to total protein.

Article Snippet: VEGF ELISA was performed with mouse VEGF polyclonal antibodies Duo-Set mouse VEGF (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions with the following modifications: The ELISA signal was detected using SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) and luminescence measured using a POLARstar luminometer (BMG LABTECH Inc., Cary, NC).

Techniques:

Journal: PLoS ONE

Article Title: A Systematic Analysis of the Peripheral and CNS Effects of Systemic LPS, IL-1Β, TNF-α and IL-6 Challenges in C57BL/6 Mice

doi: 10.1371/journal.pone.0069123

Figure Lengend Snippet: Quantitative PCR primer and probe sequences.

Article Snippet: Primary antibodies were used as follows: mouse anti-p65 (1/1000, Santa Cruz), goat anti-COX2 (1/1000, Santa Cruz), goat anti-CCL2 capture antibody (1/1000, R&D Systems CCL2 duo-set ELISA antibody pair), goat anti-CXCL1 (1/50, R&D Systems), mouse anti-cFOS (1/250, Santa Cruz), rabbit anti-IL-1β (1/50 in 20% normal goat serum, Peprotech) and goat anti-TNF-α capture antibody (1/1000, R&D Systems ELISA kit).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Amplification

Journal: PLoS ONE

Article Title: A Systematic Analysis of the Peripheral and CNS Effects of Systemic LPS, IL-1Β, TNF-α and IL-6 Challenges in C57BL/6 Mice

doi: 10.1371/journal.pone.0069123

Figure Lengend Snippet: Liver transcription of mRNA species for IL-1β, TNF-α, CXCL1, CCL2, SAA2 and TNF-αIP2 were measured using quantitative PCR in C57BL/6 female mice 2 hours after systemic challenge (i.p.) with saline, LPS (100 µg/kg), IL-1β (15 µg/kg or 50 µg/kg), TNF-α (50 µg/kg or 250 µg/kg) and IL-6 (50 µg/kg or 125 µg/kg). Data were analysed by one-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between saline, LPS and each cytokine treatment at the higher dose. ##, denotes treatment is significantly different to saline treatment # # p<0.01, # # # p<0.001. * denotes significant difference between treatment groups indicated by line p < 0.05, **p<0.01, ***p<0.001. +++ denotes significant difference between low and high dose of cytokine p<0.001. All data have been presented as mean ± SEM, n=5 for all groups.

Article Snippet: Primary antibodies were used as follows: mouse anti-p65 (1/1000, Santa Cruz), goat anti-COX2 (1/1000, Santa Cruz), goat anti-CCL2 capture antibody (1/1000, R&D Systems CCL2 duo-set ELISA antibody pair), goat anti-CXCL1 (1/50, R&D Systems), mouse anti-cFOS (1/250, Santa Cruz), rabbit anti-IL-1β (1/50 in 20% normal goat serum, Peprotech) and goat anti-TNF-α capture antibody (1/1000, R&D Systems ELISA kit).

Techniques: Real-time Polymerase Chain Reaction, Saline

Journal: PLoS ONE

Article Title: A Systematic Analysis of the Peripheral and CNS Effects of Systemic LPS, IL-1Β, TNF-α and IL-6 Challenges in C57BL/6 Mice

doi: 10.1371/journal.pone.0069123

Figure Lengend Snippet: Hippocampal mRNA transcription of CCL2, CXCL1, PTX3 and uPAR was measured using quantitative PCR after systemic challenge (i.p.) with saline, IL-1β (15 µg/kg) or TNF-α at 1, 2, 4, 8 and 24 hours post-injection. All data groups were compared by two-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between each IL-1β or TNF-α group and the equivalent saline groups and comparing the cytokines directly. # denotes treatment is significantly different to saline, while * denotes a difference between IL-1β and TNF-α. #/* p < 0.05, # #/** p < 0.01, # # #/*** p<0.001. All data have been presented as mean±SEM, n=5 for all IL-1β/TNF-α groups, except all 2 hour groups (n=4). All saline groups n≥4 except 4 and 24 hours n≥3.

Article Snippet: Primary antibodies were used as follows: mouse anti-p65 (1/1000, Santa Cruz), goat anti-COX2 (1/1000, Santa Cruz), goat anti-CCL2 capture antibody (1/1000, R&D Systems CCL2 duo-set ELISA antibody pair), goat anti-CXCL1 (1/50, R&D Systems), mouse anti-cFOS (1/250, Santa Cruz), rabbit anti-IL-1β (1/50 in 20% normal goat serum, Peprotech) and goat anti-TNF-α capture antibody (1/1000, R&D Systems ELISA kit).

Techniques: Real-time Polymerase Chain Reaction, Saline, Injection

Journal: PLoS ONE

Article Title: A Systematic Analysis of the Peripheral and CNS Effects of Systemic LPS, IL-1Β, TNF-α and IL-6 Challenges in C57BL/6 Mice

doi: 10.1371/journal.pone.0069123

Figure Lengend Snippet: Representative micrographs of vascular NFκB p65, COX-2, CCL2 and CXCL1 2h after i.p. challenge with either saline, LPS (100µg/kg), IL-1β (15µg/kg) or TNF-α (50µg/kg). All pictures were taken from the hippocampus, except for COX-2, which were taken from the thalamus since neuropil labelling with COX-2 is also high in the hippocampus, thus reducing the contrast with activated endothelium. Scale bars are 20µm and 50µm as shown.

Article Snippet: Primary antibodies were used as follows: mouse anti-p65 (1/1000, Santa Cruz), goat anti-COX2 (1/1000, Santa Cruz), goat anti-CCL2 capture antibody (1/1000, R&D Systems CCL2 duo-set ELISA antibody pair), goat anti-CXCL1 (1/50, R&D Systems), mouse anti-cFOS (1/250, Santa Cruz), rabbit anti-IL-1β (1/50 in 20% normal goat serum, Peprotech) and goat anti-TNF-α capture antibody (1/1000, R&D Systems ELISA kit).

Techniques: Saline

Journal: British Journal of Cancer

Article Title: A potential role for Dkk-1 in the pathogenesis of osteosarcoma predicts novel diagnostic and treatment strategies

doi: 10.1038/sj.bjc.6604069

Figure Lengend Snippet: ( A ) Osteogenic differentiation of MSCs in the presence of Dkk-1. Results from cells derived from three human donors and pooled murine donors are presented. Osteogenic differentiation is presented as a function of membrane ALP activity, an early marker of osteogenesis. Measurements are normalised to control levels of activity, designated 1.0. The black lines represent MSCs prepared from the fluid component of bone marrow, and the red lines represent MSCs prepared from bone spicules filtered from the aspirates. Dkk-1 exposure causes a dose-dependent inhibition of alkaline phosphatase activity. ( B ) Alizarin Red stained, long-term cultures of osteogenic MSCs in the presence and absence of Dkk-1. Calcium detection by Alizarin Red S demonstrates that Dkk-1 inhibits mineralisation of the cultures. ( C ) Immunodepletion of Dkk-1 from MG63 OS conditioned medium through incubation with a polyclonal antibody against Dkk-1. The Dkk-1–antibody complexes were removed from the medium by protein A affinity chromatography, then the medium was assayed by ELISA. ( D ) Osteogenic differentiation by MSCs in the presence of nondepleted and Dkk-1 immunodepleted conditioned medium from MG63 OS cells. Representative results from one out of three donors are presented. Measurements were achieved by ALP assay, values represent the mean ( n =6), and error bars represent s.d. P -values were calculated by two-tailed Student's t -test. ( E ) Osteogenic differentiation by MSCs in the presence of Dkk-1 and with or without the GSK3 β inhibitor BIO. ( F ) The effect of a range of BIO doses on the proliferation of OS cells. Cell numbers were evaluated by fluorescent nucleic acid intercalation assay.

Article Snippet: Enzyme-linked immunosorbent assays (ELISAs) were performed using a polyclonal duo set (R&D Systems, Minneapolis, MN, USA, catalogue no. AF1096) consisting of a goat anti-human Dkk-1 antibody and a biotinylated sample of the same serum.

Techniques: Derivative Assay, Membrane, Activity Assay, Marker, Control, Inhibition, Staining, Immunodepletion, Incubation, Affinity Chromatography, Enzyme-linked Immunosorbent Assay, ALP Assay, Two Tailed Test