elisa reagents and protocols  (LINCO)

Journal: Cells

Article Title: Aryl Hydrocarbon Receptor Activation by Benzo[ a ]pyrene Prevents Development of Septic Shock and Fatal Outcome in a Mouse Model of Systemic Salmonella enterica Infection

doi: 10.3390/cells11040737

Figure Lengend Snippet: Comparison of BaP- and FICZ-induced effects on mRNA ( a – c ) and protein expression ( d – f ) of Il1b /IL-1β, Tnfa /TNF-α and Il10 /IL-10 in peritoneal cells at days 1 and 3 post infection. Mice were treated with corn oil (vehicle control), 0.02 µg/kg or 2 µg/kg body weight BaP or 1.25 µg/kg body weight FICZ (both substances dissolved in corn oil) via i.p. injection. Two days after first application of BaP, FICZ or vehicle alone, animals were systemically infected with salmonellae by i.p. injection of 5 × 10 7 CFU of a live attenuated vaccine strain of S. enterica Serovar Enteritidis (S.E.). Changes in gene transcription were assessed by qRT-PCR. Results are expressed as BaP- or FICZ-induced relative mRNA expression compared to vehicle control (upper panel, a – c ). ∆ ct values from test and control samples were normalized against the expression of two non-regulated housekeeping genes, Alas1 and Hprt , and expressed in % of the transcription rate of the housekeeping genes. Data are shown as boxplots indicating the median and 25% and 75% confidence intervals, n = 5–6 mice per group. Protein concentration was analyzed in peritoneal cavity fluid by cytometric bead array (lower panel, d – f ). Plotted data represent mean ± SD, n = 5–14 mice per group. Differences between BaP- or FICZ-treated groups and the vehicle control were proved for statistical significance by Kruskal–Wallis one-way ANOVA or ANOVA on ranks followed by Holm–Sidak or Dunn’s post-hoc analysis (* p ≤ 0.05, ** p ≤ 0.01). n.d. not determined.

Article Snippet: For determination of IL-1β, IL-6, IL-10, IL-23, IFN-γ and TNF-α ELISA reagent sets and protocols were purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Comparison, Expressing, Infection, Injection, Quantitative RT-PCR, Protein Concentration

Journal: Cells

Article Title: Aryl Hydrocarbon Receptor Activation by Benzo[ a ]pyrene Prevents Development of Septic Shock and Fatal Outcome in a Mouse Model of Systemic Salmonella enterica Infection

doi: 10.3390/cells11040737

Figure Lengend Snippet: Effects of BaP on the systemic cytokine response (spleen) at days 3 and 10 p.i. Mice were treated with vehicle (corn oil), 0.02 µg/kg or 2 µg/kg body weight BaP (dissolved in corn oil) via i.p. injection. Two days after first application of BaP or vehicle, animals were systemically infected with salmonellae by i.p. injection of 5 × 10 7 CFU of a live attenuated vaccine strain of S. enterica Serovar Enteritidis (S.E.). On days 3 and 10 p.i. sera were generated, and concentrations of relevant proinflammatory (i.e., TNF-α, IL-6, IL-12, IFN-γ ( a – d )) and anti-inflammatory cytokines (i.e., IL-4, IL-10; ( e , f ) were analyzed by cytometric bead array (CBA)). Plotted data represent the mean values ± SD. Differences between BaP-treated groups and the vehicle control were tested for statistical significance by Kruskal–Wallis one-way ANOVA or ANOVA on ranks (if normality and/or equal variance tests failed) followed by post-hoc analysis with the Holm–Sidak method or the Dunn’s test, respectively (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: For determination of IL-1β, IL-6, IL-10, IL-23, IFN-γ and TNF-α ELISA reagent sets and protocols were purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Injection, Infection, Generated

Journal: Cells

Article Title: Aryl Hydrocarbon Receptor Activation by Benzo[ a ]pyrene Prevents Development of Septic Shock and Fatal Outcome in a Mouse Model of Systemic Salmonella enterica Infection

doi: 10.3390/cells11040737

Figure Lengend Snippet: AhR-dependent effects of BaP on the local and systemic cytokine response and MHC class II surface expression by peritoneal cavity cells (upper panel) or spleen cells (lower panel), respectively. Ahr wild-type ( Ahr +/+ ) and Ahr -deficient mice ( Ahr −/− ) were intraperitoneally injected with 2 µg/kg BaP or vehicle (corn oil). After 20 h of BaP exposure in vivo, white cells from peritoneal cavity and spleen were isolated, washed three times in PBS and cultured in RPMI1640 medium (10% FCS) at 37 °C, 5% CO 2 , 96% humidity in the presence of 2 × 10 7 CFU hkS.E. for 20 h ex vivo. Then, culture supernatants were harvested for cytokine analysis. Concentrations of IL-10 ( a , b ), TNF-α ( c ) and IL-23 ( d ) were determined by ELISA. Cells were harvested and immunophenotyped for CD11b, F4/80 and MHC II (I-A b ) expression. Shown is the mean fluorescence intensity (MFI) of I-A b staining on CD11b high /F4/80 + cells ( e , f ). Plotted data represent the mean values ± SEM (n = 4). Differences between BaP treatment and vehicle control or between Ahr +/+ and Ahr −/− mice were tested for statistical significance by Student’s t -test if the normality and equal variance test passed or by the Mann–Whitney rank sum test ( u -test) if the normality and/or equal variance test failed (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: For determination of IL-1β, IL-6, IL-10, IL-23, IFN-γ and TNF-α ELISA reagent sets and protocols were purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Injection, In Vivo, Isolation, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay, Fluorescence, Staining, MANN-WHITNEY