2.6 Bone marrow derived macrophages isolation, culture and treatment
Bone marrow derived macrophages (BMDMs) were prepared as described (Zhao et al. , 2018). Briefly, BMDMs were isolated from the male C57BL/6J mice at the age of 9 or 10 weeks, and cultured in HG-DMEM supplemented with 10% FBS and mM-CSF (20 ng/mL), and the medium was replaced on the 4th day. Cells were maintained for 7 days to differentiate into mature macrophages. The mature BMDMs were treated with LPS alone (100 ng/mL) or along with 270 for 24 h, the medium was used for ELISA test, and cells were used for RNA extraction. The mature BMDMs were pretreated with 270 for 4 h before LPS (100 ng/mL) stimulation for 30 min, and cells lysates were obtained for western blot. In the process, mM-CSF (20 ng/mL) needed to be added to the HG-DMEM containing 10% FBS to maintain the mature state of BMDMs.
2.7 RNA isolation and quantitative RT-PCR
Total RNA was isolated from cells and tissues (20 mg) using TRIzol reagent (Invitrogen). The cDNA was prepared using PrimeScript reverse transcriptase (TaKaRa) according to the manufacturer’s instructions. After a 10-fold dilution, the cDNAs were amplified using 2×SYBR Green qPCR master mix (Biotool) and a Stratagene Mx3005P (Agilent Technologies). The primers sequences were used in the paper showed in the supplementary information Table 1.