Quantstudio design and analysis desktop software

Total RNA was extracted from cells using RNeasy Protect Cell Mini Kit (Qiagen) with QIAshredder homogenizers (Qiagen). RNA was treated with DNase I (PrimerDesign or Invitrogen) according to manufacturer’s instruction, samples’ concentration was adjusted to 50 ng/µl and verified on Qubit Fluorometer (Invitrogen) using Qubit RNA HS Assay Kit (Invitrogen). Expression of DHRS9 and Ido1 mRNA was measured by RT-qPCR relative to GAPDH mRNA endogenous control using TaqPath 1-Step Multiplex Master Mix Kit (Applied Biosystems) and QuantStudio 5 Real-time PCR system (Applied Biosystems) according to manufacturer’s instruction. TaqMan assays were from Applied Biosystems: for Human Ido-1 assay number Hs00984148_m1 (FAM-MGB), for Human DHRS9 Hs00608375_m1 (FAM-MGB) and for Human GAPDH Hs03929097_g1 (VIC-MGB). Each amplification reaction contained 50 ng of RNA and was performed in triplicates. Data was analyzed with QuantStudio Design and Analysis desktop Software (Applied Biosystems). Changes in expression (Rq) were calculated relative to expression of corresponding mRNA in the starting material, i.e. CD14 + monocytes.

Total RNA was extracted from cells with TRIzol Reagent (Invitrogen, USA) according to the manufacturer's protocols. cDNA was synthesized using a Genomic reverse transcription kit (TOYOBO, Japan), and real‐time PCR was performed with QuantStudio 5 (Applied Biosystems, USA) using 2×SYBR Green qPCR Master Mix (Biomake, USA). All samples were run at least three independent experiments, and the relative gene expression was normalized to internal control cyclophilin. The expression level of each target gene was displayed by the ratio of the CTL group by Quant‐Studio Design and Analysis desktop software (Applied Biosystems, USA). The sequence of primers used for qPCR will be provided under request.
Quantitative polymerase chain reaction (qPCR) arrays are performed to analyze a panel of cytokine‐ and chemokine‐related gene expression in IL10‐eM and M‐Vec macrophages following the instructions of the manufacturer (Wcgene Biotechnology Corporation, China). The results were analyzed using software from Wcgene Biotech.

RNA was extracted using Quick‐RNA MiniPrep Plus (Zymo Research). Reverse transcription was performed using a PCR thermal cycler (Takara). Optical 96‐well reaction plates (Thermo Fisher Scientific) and optical adhesive films (Thermo Fisher Scientific) were used for the PCR. The PCR mixture loaded in each well had a final volume of 20 µL, and included 8 µL FastStart Universal SYBR Green Master Mix (Rox), 10 µL RNase‐free water, 1 µL template cDNA, and 1 µL primer. PCR amplification was conducted with the following cycling parameters: 15 min at 95 °C (heat activation step), followed by 40 cycles of 15 s at 95 °C and 1 h at 60 °C. Data were analyzed using QuantStudio Design and Analysis desktop software (Thermo Fisher Scientific). Differences in gene expression levels among different groups were statistically analyzed. Table1 shows the primer sequences. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) served as the internal control.