Foxp3 mm00475162

Liver specimens were snap-frozen in nitrogen-cooled methylene butane and then stored in liquid nitrogen until RNA analysis. Total RNA was isolated (RNeasy Plus Mini Kit, Qiagen, Germany) and reversely transcribed into cDNA using High-capacity cDNA Reverse Transcriptase Kit (ThermoFisher, Germany) according to the manufacturer’s instructions. Real-time PCR (RT-PCR) was performed in triplicates using the following TaqMan Gene Expression Assays: Foxp3 Mm00475162; Ctla4 Mm00486849; Arg1 Mm00475988; Retnla Mm00445109 (ThermoFisher, Germany). The reaction was performed on the 7900HT Fast Real-Time PCR System under the following reaction conditions: thermal cycling conditions were 50°C for 2 minutes followed by 95°C for 10 minutes, 45 cycles at 95°C for 15 seconds, and at 60°C for 1 minute. Gene expression values were normalized to the endogenous reference gene GAPDH (Rodent GAPDH control reagent, ThermoFisher, Germany) and presented as normalized expression values relative to naive controls.

Six weeks after infection, total RNA was isolated from snap-frozen spleens (RNeasy Plus Mini Kit, Qiagen, Germany) and reversely transcribed into cDNA using High-Capacity cDNA Reverse Transcriptase Kit (ThermoFisher, Germany) according to the manufacturer’s instructions. RT-PCR was performed using the following TaqMan Gene Expression Assays: Foxp3 Mm00475162; Il10 Mm01288386, Ctla4 Mm00486849; Rnf138 Mm01158179; Il33 Mm00505403 and Il1rl1 Mm00516117 (ThermoFisher, Germany). Cycling was performed on the 7900HT Fast RT-PCR System under the following reaction conditions: 50°C for 2 min followed by 95°C for 10 min, 45 cycles at 95°C for 15 s, and at 60°C for 1 min. Gene expression values were normalized to the endogenous reference gene GAPDH (Rodent GAPDH control reagent, ThermoFisher, Germany) and presented as normalized, expression values relative to naive controls.