Ultrapure rnase free water

Changes in the mRNA expression profiles were monitored as reported in our previous studies [11 (link),42 (link),46 (link)]. Briefly, moMΦ were seeded in 12-well plates and left untreated or stimulated with recombinant IL-10 or recombinant TGF-β (both at 20 ng/mL); at 4 and 24 h post-treatment, culture supernatants were discarded, and moMΦ were lysed with buffer RTL (Qiagen, Hilden, Germany). Total RNA was then extracted using the RNeasy Mini Kit, treated with RNase-Free DNase Set, and finally eluted in 100 µL of ultrapure RNase-free water (all Qiagen, Milan, Italy). Then, 250 ng of purified RNA was used as a template for cDNA synthesis [13 (link),42 (link),46 (link)]. RT-qPCR was performed to evaluate gene expression, using the primer sets reported in Table S1. Real-time PCR amplification was performed in a CFX96™ Real-Time System after the reverse transcription step, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene [45 (link)]. In each sample, the relative expression of the test genes was calculated from Cq (quantification cycle) values, using the widely adopted 2^−ΔΔCq method [13 (link),42 (link),46 (link)].

Changes in the mRNA expression profiles were monitored as previously described [14 (link)]. In brief, moMΦ were seeded in 12-well plates and either left untreated or stimulated with a variety of TLR2 agonists (all at 100 ng/mL); moM1 (IFN-γ/LPS) were also included in the experiment. After 24 h, culture supernatants were removed and cells were lysed using buffer RTL (Qiagen, Hilden, Germany). Then, total RNA was extracted using the RNeasy Mini Kit, treated with the Rnase-Free Dnase Set, and eluted in 50 µL of ultrapure Rnase-free water (all from Qiagen, Milan, Italy). An amount of 250 ng of the obtained purified RNA was used as a template for cDNA synthesis [14 (link)]. Then, RT-qPCR was performed to evaluate expression of several genes of the innate immune system (IL-1β, IL-6, IL-10, IL-12p40, TNF-α, IFN-β, TLR2, TLR3, TLR7, TLR8, TLR9, cGAS, STING, RIG-I, MDA5, and IRF3), using the primer sets reported in the Supplementary Table S3 [14 (link),33 (link),34 (link),35 (link),36 (link),37 (link)]. Real-time PCR amplification was performed in a CFX96™ Real-Time System after the reverse transcription step; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene [14 (link)]. In each sample, the relative expression of the tested genes was calculated from Cq (quantification cycle) values using the widely adopted 2^−ΔΔCq method [14 (link)].