Perfect real time supporting system

Ileal tissues were washed with cold phosphate-buffered saline and stored in RNAlater (Ambion, Austin, TX, USA) at 4 °C until use. Total RNA was extracted using Sepasol-RNA I Super G (Nacalai Tesque) according to the manufacturer's instructions and reverse-transcribed using PrimeScript Reverse Transcriptase (Takara Bio, Shiga, Japan). Expression of cytokines was quantified using a Thermal Cycler Dice Real Time System (Takara Bio) with SYBR Premix ExTaq II (Takara). Pre-designed primer sets for mouse TNF-α (primer set MA097070), IL-6 (primer set MA039013), IFN-γ (MA025911), IL-17 (MA157056), and TATA-binding protein (TBP) (MA050367) were obtained from the Perfect Real-Time Supporting System (Takara Bio). The mRNA expression level was standardised to that of TBP.

Mouse monocyte/macrophage cell line RAW264 were obtained from RIKEN BRC (Ibaraki, Japan). The cells were grown in Dulbecco's modified Eagle medium (DMEM: Wako) containing 10% fetal bovine serum (FBS) with 100 U/mL of penicillin G and 100 µg/mL of streptomycin at 37 ˚C in 5% CO 2 with a humidified atmosphere. Sub-confluent cells in 12-well plate were exposed of lipopolysaccharide (LPS) (Sigma-Aldrich) at a concentration of 1 µg/mL and total RNA was extracted 4 h later. Subsequently reverse transcription and PCR were performed as described above. Pre-designed primer sets for mouse TNF-α (primer set MA097070), IL-6 (primer set MA039013), IL-12A (MA0287533), IL-23A (MA095159), and TATA-binding protein (TBP) (MA050367) were obtained from the Perfect Real-Time Supporting System (Takara Bio). The mRNA expression level was standardised to that of TBP.
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted January 24, 2020. ; https://doi.org/10.1101/2020.01.24.917948 doi: bioRxiv preprint CN210 at 0.1, 1 and 10 μM and dexamethasone at 1 μM were co-treated with LPS in RAW264 cells.