For eight weeks. Cell fluorescence in each replicate was measured having a cytometer (BD FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA) just after 24, 48, 72, and 96 h of exposure, and weekly thereafter. For cytometer measurements, 1 105 cells of each replicate have been diluted in 500 of PBS. Each sample was analyzed for the percentage of fluorescent cells present, as well because the relative fluorescence intensity of every single sample. The experiment was carried out in conjunction with unexposed time-matched control cells. 2.8. Real-Time RT CR Gene Expression Evaluation The expression of the oxidative-damage (HO1, SOD2, GSTP-1) and general-stress (HSP70) associated genes was analyzed by Real-Time RT CR just after short and long-term exposure of Caco-2 cells to PSNPs. Additionally, ACTB was made use of because the housekeeping reference gene. Cells have been exposed to PSNPs for 24 h (quick term) or eight weeks (long-term). Both for short- and long-term exposed cells, RNA extraction was carried out making use of TRI Reagent(Invitrogen, Waltham, MA, USA), in accordance with the product’s recommended protocol. Extracted RNA samples were then treated with RNase-free DNAse I (Turbo DNA-free kit; Invitrogen, USA) for 1 h and quantified utilizing Nanodrop (Nanodrop Spectrophotometer ND-1000). Retrotranscription was carried out employing 2000 ng of RNA per sample with all the High-Capacity RNA-to-cDNA kit (Applied Biosystems, M50054 site Bedford, MA, USA), along with the level of cDNA after retrotranscription was quantified in every sample, again applying Nanodrop (Nanodrop Spectrophotometer ND-1000). Samples had been then diluted in RNase-free water to attain a final concentration of ten 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. Every 20 of reaction volume contained five of cDNA (50 ng of cDNA), ten of 2LightCycler-480 SYBR Green I Master (Roche, Switzerland), 3 of distilled water, and 1 of every single primer (forward and reverse) at a final concentration of 10 . The primer sequences utilized will be the following: HO1 F: 5 -TCCGATGGGTCCTTACACTC-3 , R: five -AAGGAAGCCAGCCAAGAGA-3 ; GSTP1 F: 5′-CCAATACCATCCTGCGTCAC-3 , R: 5 -CAGCAAGTCCAGCAGGTTGT-3 ; HSP70 F: five -TGATCAACGACGGAGACAAG-3 , R: 5 -TCCTTCATCTTGGTCAGCAC-3 ; SOD2 F: five -GGCCTACGTGAACAACCTGA-3 , R: five -GAGCCTTGGACACCAACAGA-3 ; ACTB F: five -GCATGGAGTCCTGTGGCATC-3 , R: five -CCACACGGAGTACTTGCGCT-3 . 3 wells per replicate, concentration, and target gene had been applied. The LightCycler-480 parameters were as follows: pre-incubation at 95 C for 5 min; 45 cycles of 95 C for 10 s; 62 C for 15 s; and 72 C for 25 s. The data around the o-Toluic acid supplier crossing points (Cp) for every single sample was obtained utilizing the LightCycler-480 software. Target gene values were 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 damage was evaluated in Caco-2 cells immediately after 24 h and 8 weeks of exposure to various concentrations of PSNPs. In addition to, negative and constructive controls were set up. Constructive controls were treated with five mM KBrO3 , and 200 MMS forBiomolecules 2021, 11,five 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 had been resuspended in PBS to attain a dilution of 106 cells per mL. Subsequently, every single sample was mixed with previously heated agar, the mi.