materials raw blue selection antibiotic zeocin  (InvivoGen)

Journal: Biochemical pharmacology

Article Title: Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage

doi: 10.1016/j.bcp.2017.11.012

Figure Lengend Snippet: (A) Transcriptional expression of TLR4 mRNA in RAW-Blue cells by RT-qPCR. Primary peritoneal macrophages (pM) isolated from TLR4-WT and TLR4-KO mice were used as positive and negative control, respectively. B) Comparative TEB gel electrophoresis of TLR4 real time PCR products. (C) TLR4 expression in RAW-Blue cells and pM from TLR4-KO mice following subjection of cell lysates to Western blot analysis.

Article Snippet: 2.1 Chemicals and Materials RAW-Blue selection antibiotic Zeocin, Quanti-Blue detection reagent (alkaline phosphatase detection medium), LPS-EK from E. coli K12 (LPS-EK Ultrapure), and TLR4 signaling inhibitor CLI-095 were obtained from InvivoGen (San Diego, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Negative Control, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biochemical pharmacology

Article Title: Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage

doi: 10.1016/j.bcp.2017.11.012

Figure Lengend Snippet: RAW-Blue cells were plated at 5 × 105 cells/well in 24-well plates. Cells were treated with oxidants or LPS-EK at the indicated concentrations for 2 h. Media was collected and cells rinsed in fresh media followed by re-incubation with fresh medium in the absence of oxidants or LPS-EK for the following 16 h. The cytotoxicity resulting from treatment with PPC, SIN-1 or LPS-EK at indicated concentrations were determined by quantification of LDH released into culture medium at the end of 2 h (A) and 16 h incubation (B). Cell viabilities upon different treatments were also determined by live cell counting with trypan blue staining at 2 h (C) and 16 h (D) for cell proliferation assay. Cell # was counted at the end of 16 h treatment. The white (blank) bars represent vehicle control, while the black bars represent the initial cell seeding number of 5 × 105 cells. The data represent 3 independent experiments conducted in duplicate at different times. # p ≤ 0.01 vs initial seeding number; * p ≤ 0.01 vs untreated cells. 1-way ANOVA followed Tukey’s post hoc tests.

Article Snippet: 2.1 Chemicals and Materials RAW-Blue selection antibiotic Zeocin, Quanti-Blue detection reagent (alkaline phosphatase detection medium), LPS-EK from E. coli K12 (LPS-EK Ultrapure), and TLR4 signaling inhibitor CLI-095 were obtained from InvivoGen (San Diego, CA, USA).

Techniques: Incubation, Cell Counting, Staining, Proliferation Assay

Journal: Biochemical pharmacology

Article Title: Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage

doi: 10.1016/j.bcp.2017.11.012

Figure Lengend Snippet: Cells were exposed to oxidants or LPS-EK at the indicated concentrations for 2 h followed by incubation with complete growth medium (oxidants or LPS-EK free) for next 16 h. Anti-TLR4/MD2 pAb (20 μg/ml) or CLI-095 (5 μM) was added 3 h before oxidants or LPS-EK stimulation. SEAP released in the culture medium at the end of 16 h incubation was determined using Quanti-Blue assay as per the manufacturer’s instructions, and the absorbance was read at 650 nm. (A) PPC concentration-dependent SEAP release. The data represent 6 independent experiments carried out in triplicate. *p ≤ 0.005 vs control; +p ≤ 0.05 vs control. (B) SIN-1 stimulated SEAP release in a dose-dependent manner. The data represent 6 independent experiments carried out in triplicate. +p ≤ 0.05 vs control, *p ≤ 0.005 vs control. (C) Pretreatment with anti-oxidant reagent EUK-134 (4 μM) inhibited PPC or SIN-1 induced SEAP release. The data represent 4 independent experiments carried out in triplicate. *p ≤ 0.005 vs treatment with PPC or SIN-1 alone. (D) Pretreatment with Anti-TLR4 pAb or CLI-095 reduced oxidants or LPS-EK induced SEAP release in RAW-Blue cells. The data represent 4 independent experiments carried out in triplicate. *p ≤ 0.05, **p ≤ 0.005 vs treatment with PPC alone; # p ≤ 0.05 vs SIN-1 treatment alone; +p ≤ 0.05 vs LPS-EK treatment alone, ++p ≤ 0.005 vs LPS-EK treatment alone, 1-way ANOVA in all cases followed by Tukey’s post-hoc tests.

Article Snippet: 2.1 Chemicals and Materials RAW-Blue selection antibiotic Zeocin, Quanti-Blue detection reagent (alkaline phosphatase detection medium), LPS-EK from E. coli K12 (LPS-EK Ultrapure), and TLR4 signaling inhibitor CLI-095 were obtained from InvivoGen (San Diego, CA, USA).

Techniques: Incubation, Concentration Assay