Tbx21

Cells were lysed in RLT buffer (Qiagen) containing 1% 2-mercaptoethanol and subsequently homogenized using the QIAshredder (Qiagen). The total RNA was obtained with the RNeasy® mini/micro kit (Qiagen), whereas the complementary DNA was generated using a SuperScript™ VILO cDNA Synthesis Kit (Invitrogen) and used as a template for quantitative RT-PCR performed with the Fast Start Essential DNA Green Master kit (Roche). Primers for TBX21, GATA3, RORC, FOXP3, MAF, and AHR were purchased from Qiagen. A primer for ACTB was designed as follows: forward 5′-CACTCTTCCAGCCTTCCTTCC-3′, reverse 5′-GCATACAGGTCTTTGCGGATG-3′.

RNA was converted to cDNA using the RT² first strand kit (QIAGEN) as per the manufacturer's instructions. Quantitative real‐time PCR reactions were carried out on a Rotor‐Gene Q (QIAGEN) using cDNA from the above reaction, RT2 SYBR Green qPCR Mastermix (QIAGEN) and primers RPLP0 (housekeeping gene, Cat PPH21138F) IL‐4 (CAT PPH00565B), IL‐13 (CAT PPH00688F), TNF (CAT PPH00341F), IFNγ (CAT PPH00380C), RORC (CAT PPH05877A), TBX21 (Cat PPH00396A) and GATA3 (CAT PPH02143A, all purchased as commercial primers (QIAGEN). Reaction wells each contained 20 μL of reaction mix with cycling conditions as per the manufacturer's instructions. Each qPCR reaction was tested in triplicated for each gene. Fold change was expressed relative to the housekeeper gene (which was reliably detected in each experiment) and determined by the calculation 2^−ΔCT.