Total RNA from stimulated murine cells was isolated with the RNeasy Mini kit (Qiagen, Hilden, Germany), and cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). TaqMan gene expression assays (IFN-γ Mm01168134 _m1; Rn18S Mm03928990_g1), TaqMan® Fast Universal PCR Master Mix 10 × 250 Rxn and a StepOne Plus instrument (all Applied Biosystems) were used for real-time detection of IFN-γ target gene complementary DNA amplification. Ribosomal 18S was used as endogenous reference. Relative expression was calculated by the 2−ΔΔCt method.

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