Effects of heat treatment on the number of TNF-α producing cells and LPS-induced TNF-α gene expression.

<p><i>A</i> to <i>C</i>, Peritoneal macrophages were isolated 2 hours post injection from LPS-challenged mice with or without HT, recovered overnight and re-stimulated with LPS and IFN-γ at 37°C for 24 hour. TNF-α producing cell was measured by ELISpot assay. The absolute number of TNF-α-producing macrophages was calculated per 10⁴ macrophages (<i>B</i>) or calculated based on different sizes of spot area (<i>C</i>) by the Carl Zeiss Vision EliSPOT software. Data are mean ± SD. Cells from each treatment condition were pooled from 4 mice. Data are representative of two independent experiments. *<i>p</i><0.05; paired Student <i>t</i> test. <i>D</i>, Peritoneal macrophages were isolated from LPS-challenged mice as mentioned before, allowed to recover overnight and then re-stimulated (1×10⁶/well) with LPS and IFN-γ at 37°C for 4 hours to measure TNF-α mRNA by quantitative real-time PCR. The results are presented relative to GAPDH and baseline expression in unstimulated cells from RT-mice. <i>E</i>, RNA decay analysis was performed by using peritoneal macrophages (1×10⁶/well) from LPS-challenged mice that were re-stimulated with LPS and IFN-γ at 37°C for 2 hours. Actinomycin D (5 µg/ml) was then added to block further transcription. Total RNA was extracted at different time points after actinomycin D blocking and then analyzed for TNF-α mRNA expression by quantitative real-time PCR. Data represents the percent change of TNF-α mRNA compared to the cells before actinomycin D treatment. Data are measured in duplicate by Q-PCR.