Legendplex mouse t helper cytokine panel

For RNA isolation, the RNeasy kit (Qiagen) was used for FACS-sorted cells; otherwise, Trizol was used. cDNA was analyzed by real-time PCR with KAPA SYBR FAST qPCR master mix (KK4605) with the primers shown in Table S1. Gene-expression levels were evaluated by the −ΔΔCt method with Actb as an internal control. In Figures 4H, S3H, and S7A, error bars were calculated as RQ-Min = 2^{−(ΔΔCt + T*SD (ΔCt))} and RQ-Max = 2^{−(ΔΔCt − T*SD(ΔCt))} from triplicate wells as suggested by a manufacturer of PCR machines (Applied Biosystems). T*SD(ΔCt) is a square root of x²+y², where x and y are standard deviations of Ct values for a gene of interest and internal control, respectively. RQ-MIN and RQ-MAX are the acceptable errors for a 95% confidence limit according to Student’s t-test. The results shown are representative of multiple independent experiments with similar results. To evaluate cytokine serum concentrations, LEGENDplex mouse T helper cytokine panel (BioLegend) was used. sOPN ELISA was performed as previously described (Shinohara et al., 2005 (link)).