For 8 weeks. Cell fluorescence in every single replicate was measured with a cytometer (BD FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA) following 24, 48, 72, and 96 h of exposure, and weekly thereafter. For cytometer measurements, 1 105 cells of each and every replicate had been diluted in 500 of PBS. Every N-Hexanoyl-L-homoserine lactone medchemexpress sample was analyzed for the percentage of fluorescent cells present, also because the relative fluorescence intensity of every sample. The experiment was carried out in conjunction with unexposed time-matched handle cells. two.8. Real-Time RT CR Gene Expression Evaluation The expression on the oxidative-damage (HO1, SOD2, GSTP-1) and general-stress (HSP70) associated genes was analyzed by Real-Time RT CR soon after quick and long-term exposure of Caco-2 cells to PSNPs. Furthermore, ACTB was employed as the housekeeping reference gene. Cells were exposed to PSNPs for 24 h (quick term) or eight weeks (long term). Each for short- and long-term exposed cells, RNA extraction was carried out using TRI Reagent(Invitrogen, Waltham, MA, USA), in line with the product’s recommended protocol. Extracted RNA samples had been then treated with RNase-free DNAse I (Turbo DNA-free kit; Invitrogen, USA) for 1 h and quantified working with Nanodrop (Nanodrop Spectrophotometer ND-1000). Retrotranscription was carried out making use of 2000 ng of RNA per sample using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, Bedford, MA, USA), and also the level of cDNA soon after retrotranscription was quantified in each sample, once again applying Nanodrop (Nanodrop Spectrophotometer ND-1000). Samples were then diluted in RNase-free water to attain a final concentration of 10 ng/ of cDNA. The real-time RT-PCR evaluation was then performed together with the cDNA samples on a LightCycler-480 (Roche, Basel, Switzerland) to evaluate the expression levels of the targeted genes. Each 20 of reaction volume contained five of cDNA (50 ng of cDNA), 10 of 2LightCycler-480 SYBR Green I Master (Roche, Switzerland), three of distilled water, and 1 of each primer (forward and reverse) at a final concentration of ten . The primer sequences made use of are the following: HO1 F: 5 -TCCGATGGGTCCTTACACTC-3 , R: 5 -AAGGAAGCCAGCCAAGAGA-3 ; GSTP1 F: 5′-CCAATACCATCCTGCGTCAC-3 , R: five -CAGCAAGTCCAGCAGGTTGT-3 ; HSP70 F: five -TGATCAACGACGGAGACAAG-3 , R: five -TCCTTCATCTTGGTCAGCAC-3 ; SOD2 F: five -GGCCTACGTGAACAACCTGA-3 , R: five -GAGCCTTGGACACCAACAGA-3 ; ACTB F: five -GCATGGAGTCCTGTGGCATC-3 , R: 5 -CCACACGGAGTACTTGCGCT-3 . 3 wells per replicate, concentration, and target gene were utilised. The LightCycler-480 parameters had been as follows: pre-incubation at 95 C for five min; 45 cycles of 95 C for ten s; 62 C for 15 s; and 72 C for 25 s. The information around the crossing points (Cp) for each sample was obtained employing the LightCycler-480 software. Target gene Maresin 1 site values have been normalized against the values for the housekeeping gene and analyzed statistically for significance. two.9. Genotoxic and Oxidative DNA Harm Assessment in the Comet Assay Genotoxic and oxidative DNA harm was evaluated in Caco-2 cells soon after 24 h and eight weeks of exposure to different concentrations of PSNPs. Apart from, adverse and optimistic controls were setup. Positive controls had been treated with five mM KBrO3 , and 200 MMS forBiomolecules 2021, 11,5 of30 min, as inducers of oxidative and genotoxic DNA harm, respectively. Exposed/control cells had been centrifuged at 1000 rpm for eight min and cell pellets were resuspended in PBS to attain a dilution of 106 cells per mL. Subsequently, every single sample was mixed with previously heated agar, the mi.