human timp 4 elisa kit  (Boster Bio)

Journal: JCI Insight

Article Title: Autoimmune inner ear disease patient–associated 28-kDa proinflammatory IL-1 β fragment results from caspase-7–mediated cleavage in vitro

doi: 10.1172/jci.insight.130845

Figure Lengend Snippet: Expression levels of (A) IL-1β, (B) IL-6, (C) CCL3, (D) MMP-9, (E) TIMP-1, and (F) TNF-α mRNA from PBMCs of patients with AIED (n = 9) and controls (n is shown for each panel) treated with 1 μg/mL LPS (positive control); recombinant IL-1β (rIL-1β) (Peprotech); or 17-kDa, 28-kDa, and 31-kDa (negative control) fragments of IL-1β or left untreated. The expression levels were measured in replicate samples by real-time quantitative PCR (qPCR) using 480 LightCycler. Data shown are mean ± SEM. The threshold cycle (Ct) value was calculated from amplification plots, and gene expression was normalized using the Ct of the housekeeping gene glyceraldehyde-3-phosphate. Fold induction of the target gene was calculated using the formula 2−ΔΔCt. Statistical significance was achieved for the difference in expression for IL-1β in response to the 28-kDa and 17-kDa fragments in control subjects (P = 0.016). Although differences were observed between expression in response to the 28-kDa and 17-kDa fragments for IL-6 and CCL3, these did not achieve significance. MMP-9 and TNF-α expression showed minimal, insignificant changes in response to the IL-1 fragments. The Wilcoxon signed-rank test was performed for paired observations, to compare expression levels within groups, in response to the 17-kDa and 28-kDa fragments. For the pairwise comparison of 17-kDa and 28-kDa fragments, a Bonferroni’s adjustment was made for the 2 hypothesis tests carried out within a cytokine. The 2 tests were carried out comparing 17-kDa and 28-kDa fragments within the AIED group and within the control group, such that any given comparison required P < (0.05 / 2) = 0.025. Each experiment was repeated twice to confirm reproducibility.

Article Snippet: After brief centrifugation, cell culture supernatant samples were analyzed using human IL-1β, IL-6, CCL3, TNF-α, TIMP-1, MMP-9, and IL-4 ELISA kits (all from R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Positive Control, Recombinant, Negative Control, Real-time Polymerase Chain Reaction, Amplification, Comparison

Journal: JCI Insight

Article Title: Autoimmune inner ear disease patient–associated 28-kDa proinflammatory IL-1 β fragment results from caspase-7–mediated cleavage in vitro

doi: 10.1172/jci.insight.130845

Figure Lengend Snippet: PBMCs of patients with AIED and controls (n is shown for each panel) were treated with 1 μg/mL LPS (positive control); rIL-1β; or 17-kDa, 28-kDa, and 31-kDa (negative control) fragments of IL-1β or left untreated (16 hours). Cytokines were measured by ELISA from the culture supernatants. Data represent the mean ± SEM in all panels. (A) The concentration of CCL3 was measured by ELISA from the culture supernatants. A trend was observed in control PBMCs in response to the 28-kDa fragment as compared with the 17-kDa fragment (P = 0.027). (B) The TNF-α concentration was determined using a TNF-α ELISA kit. A statistically significant difference was observed in control PBMCs in response to the 28-kDa fragment as compared with the 17-kDa fragment (P = 0.008). (C) IL-6 concentrations in culture supernatants were determined by ELISA. (D) MMP-9 levels were detected by ELISA in the culture supernatant. A trend was observed in control PBMCs in response to the 28-kDa fragment as compared with the 17-kDa fragment (P = 0.027). (E) TIMP-1 supernatant concentrations were similarly measured by ELISA. (F) To determine whether the 28-kDa fragment of IL-1β had antiinflammatory properties, PBMCs of AIED and control subjects were assayed for IL-4 release by ELISA. For A–F, the Wilcoxon signed-rank test was performed for paired observations, to compare expression levels within groups, between 17 kDa and 28 kDa. For the pairwise comparison of 17 kDa and 28 kDa, a Bonferroni’s adjustment was made for the 2 hypothesis tests carried out within a cytokine. The 2 tests were carried out comparing 17 kDa and 28 kDa within the AIED group and within the control group, such that any given comparison required P < (0.05 / 2) = 0.025. Each experiment was repeated twice to confirm reproducibility.

Article Snippet: After brief centrifugation, cell culture supernatant samples were analyzed using human IL-1β, IL-6, CCL3, TNF-α, TIMP-1, MMP-9, and IL-4 ELISA kits (all from R&D Systems) according to the manufacturer’s instructions.

Techniques: Positive Control, Negative Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Comparison