Peritoneal macrophages from LPS-challenged, heat-treated mice show more NF-κB activation after <i>in vitro</i> LPS/IFN-γ re-stimulation.

<p><i>A</i> to <i>B</i>, Peritoneal macrophages were isolated from naïve or LPS-challenged, RT or heat-treated mice, recovered overnight and stimulated (1×10⁶/well) <i>in vitro</i> with LPS/IFN-γ at 37°C for 30 min (<i>A</i>) or indicated time points (<i>B</i>), stained with antibodies against CD11b, NF-κB p65 and DRAQ5 DNA dye and then analyzed by ImageStream flow cytometry. CD11b⁺ cells were gated to show the similarity score between DRAQ5 nuclear staining and NF-κB staining. Rd value = (similarity score of treated - untreated)/(standard deviation of the similarity score from treated + untreated). <i>C</i>, Macrophages (1×10⁶/well) from LPS-challenged, RT or heat-treated mice were re-stimulated <i>in vitro</i> with LPS/IFN-γ at 37°C for 0, 5, 15 and 30 min to detect the phosphorylation of IKK and IκB by Western blotting. The graph shows the quantification of the band intensity of pIKK and pIκB normalized to β-actin. Data are mean ± SD from three independent experiments. <i>D</i>, Macrophages (1×10⁶/well) from LPS-challenged, RT or heat-treated mice were stimulated <i>in vitro</i> with LPS/IFN-γ at 37°C for 30 min. Cross-linked chromatin was immunoprecipitated with anti-NF-κB p65 antibody and analyzed for NF-κB p65 binding to the TNF-α promoter region by quantitative real-time PCR with primers spanning the regions −364/−182, −586/−468, −685/−543 and −912/−763. The graph shows the fold change which is normalized to the input and control IgG and then compared with unstimulated or RT-unstimulated cells. Cells from each treatment condition were pooled from 4 mice. Data are representative of two independent experiments.