Total RNA was isolated from the HMC3 cells with a Trizol LS Reagent Kit (Life Technologies, Milan, Italy) and then quantified with a NanoDrop Lite spectrophotometer (Thermo Fisher, Milan, Italy). A total of 1 μg of total RNA was reverse-transcribed in a final volume of 20 μL by using the Superscript IV RT Master Mix (Invitrogen, Carlsbad, CA, USA). A total of 1 μL of cDNA was added to the BrightGreen qPCR Master Mix (ABM, Richmond, BC, Canada) together with specific primers at the concentration of 10 μM in a total volume of 20 μL/well to evaluate IL-1β, IL-6, Il-10, NLRP3, Caspase-1, and TNF-α mRNA expression. A qPCR reaction was monitored by using the QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher, Milan, Italy); GAPDH was used as housekeeping gene and the amplified PCR products were quantified by measuring the cycle thresholds (CT) of the target genes and GAPDH. After normalization, the mean value of the control group was chosen as the calibrator and the results were expressed according to the 2−ΔΔCt method, as a fold change relative to the calibrator [29 (link),30 (link),31 (link),32 (link)]. Primers used for targets and reference genes are listed in Table 1.
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