2.9∣RNA isolation and real-time quantitative PCR
Total RNA was extracted from splenocytes of individual mice after stimulation with 1 μg/mL of rFor t 2 or medium alone for 3 days using TRIzol reagents. First-strand cDNA synthesis was carried out using SuperScript III kit (Invitrogen, Carlsbad, CA). The analyzed target genes were IL-10, IL-13, IFN-γ, and FOXP3, and these specific primers are given in supplementary Table 1. PCR reactions were run on an ABI StepOnePlus machine (Life Technologies) as follows: an initial 10-minute step at 95℃ followed by 40 cycles of 95℃ for 15 seconds and 60℃ for 1 minute. The fluorescent signal from SYBR Green was detected immediately after the extension step of each cycle, and the cycle at which the product was first detectable was recorded as the cycle threshold. Data were imported into an Excel database and analyzed using the comparative cycle threshold method with normalization of the raw data to β-actin.