After being removed with trypsin, the MPMs were stored with RNAlater at -70°C and, later, destined for the gene expression of inflammatory markers by the RT-qPCR array methodology. The MPMs mRNA was extracted using the TRIzol® Plus RNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA) following the protocol provided by the manufacturer. The cDNA was obtained by a SuperScript III Reverse Transcriptase Kit. We used personalized plates of the target genes IL-1β, IL-6, IL-8, TNF-α, and TLR2. The SYBR® PCR Master Mix test gene expression (Applied Biosystems™) was performed on personalized plates with targets genes, endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin genes and MGDC control genes (Mouse Genomic DNA Contamination), RTC (Reverse Control Transcription) and PPC (Positive PCR Control). Classically activated macrophages in S. aureus more commonly adopt TLR ligation signaling via MyD88 and NF-κB, generating TNF-α, IL-1β, IL-6, and IL-8 (Cole et al., 2014; Tarique et al., 2015). Classically activated macrophages also express high levels of TLR2 (Cole et al., 2014), which has been greatly implicated in host defense against S. aureus (Miller and Cho, 2011; Krishna and Miller, 2012). Amplification was performed on the StepOnePlusTM Software v2.3 thermocycler, with the following parameters for all genes: 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The data were analyzed using the comparative method (2ΔΔCt) and normalization was performed based on GAPDH expression.

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