Light cycler sybr green 1 master

After washing, adherent macrophages were immediately stimulated with IFNγ (40 UI ml⁻¹, Clinisciences), IL-6 (50 ng ml⁻¹, Clinisciences), LPS (1 ng ml⁻¹, Sigma), IL-4 (50 ng ml⁻¹, Miltenyi Biotech), IL-13 (50 ng ml⁻¹, Clinisciences), IL-10 (50 ng ml⁻¹, Clinisciences), 15-HETE (1 μM, Cayman) or DLPC (50 μM, Sigma) for 4 or 24 h. In indicated experiments, adherent macrophages were pre-incubated or not with a Jak-2/STAT6 inhibitor, AG490 (1 nM, Tebu-Bio).
The mRNA preparation was made using the EZ-10 Spin Column Total RNA Minipreps Super Kit (Bio Basic) using the manufacturer's protocol. Synthesis of cDNA was performed according to the manufacturer's recommendations (Thermo electron). RT–qPCR was performed on a LightCycler 480 system using LightCycler SYBR Green I Master (Roche Diagnostics). The primers (Eurogentec) were designed with the software Primer 3. Actb (Actin) mRNA was used as the invariant control. Serially diluted samples of pooled cDNA were used as external standards in each run for the quantification. Primer sequences are listed in Supplementary Table 1.

Total RNA was extracted using RNAprep pure MicroKit (Aidlab, Beijing, China) following the manufacturer’s instructions. cDNA was synthesized by using cDNA Synthesis Kit (TaKaRa, Dalian, China). Reverse transcription parameters were set as follows: 42 °C for 50 min and 65 °C for 15 min. For PCR, the primers were listed in table 1. PCR amplification was carried out on a Light Cycler real-time PCR instrument (Roche, Mannheim, Germany). The amplification procedure was conducted according to manufacturer’s instructions using a Light Cycler SYBR Green I Master (Roche). The PCR parameter were set at 95 °C for 10 min, followed by 55 cycles at 95 °C for 10 s, 60 °C for 30 s, and finally cooling at 4 °C.

Total RNA was extracted from cell aggregates using SPARKeasy Tissue/Cell RNA Rapid Extraction Kit (SPARKjade, AC0202, Shandong, China) and reverse transcription was performed using a SPARKScript RT plus Kit (SPARKjade, AG0304). A Light Cycler SYBR Green I Master (Roche, 04707516001, Mannheim, Germany) kit was used to prepare PCR mix and PCR was performed using a Light Cycler 480 Real-Time PCR System (Roche, Mannheim, Germany). The relative gene expression was calculated using the 2^{−(△△Ct)} method and Gaphd was used as a housekeeping gene. Primer sequences used are listed in Additional file 2: Table S2.