Corbett rotor gene thermal cycler

The analysis of the anti-inflammatory activity of the Fucopol HMs in THP1 cells proceeded as previously described with a few modifications [66 (link)]. Briefly, 1 × 10⁶ THP1 cells were seeded in 6-well plates, incubated for 2 h with 7 µg/mL Lipopolysaccharide (LPS, Sigma Aldrich, St. Louis, MO, USA) and were then incubated for 30 min or 3 h with the Fucopol HMs (13 mm diameter) or with 500 µg/mL of Fucopol. Identical samples without the addition of LPS were prepared (-LPS) for negative control purposes. In another approach, cells were simultaneously exposed for 2 h to both LPS and the Fucopol HMs or to 500 µg/mL of Fucopol. After the incubation time, cells were pelleted via centrifugation (500× g, 5 min) and solubilized in 300 µL of NZYol (NZYtech, Lisboa, Portugal), and RNA was extracted according to the manufacturer’s instructions and reverse-transcribed with the NZY M-MulV First strand cDNA synthesis kit (NZYtech). The relative expression of the tumor necrosis factor α gene (TNF-α) and of the housekeeping gene RNA18S were determined using the Ct method (2^−∆∆Ct) [67 (link)] after real-time quantitative amplification using the NZYSupreme qPCR Green Master Mix (NZYtech) in a Corbett Rotor-Gene thermal cycler (Qiagen, Hilden, Germany).

The anti-inflammatory potential of the peptide fraction 2–10 kDa was examined in coN cells with few alterations from what was previously published [78 (link)]. Fibroblasts and coN cells were seeded in 25 cm² T-flasks and then incubated for 24 h at 37 °C, 5% (v/v) CO₂ and 99% (v/v) relative humidity. Cells were incubated for 2 h with 7 µg/mL lipopolysaccharide (LPS, Sigma Aldrich, St. Louis, MO, USA), then 0.25 mg/mL peptide fraction 2–10 kDa or 1% (v/v) ethanol were added, and cells incubated for further 3 h. Similar samples without LPS were also prepared in parallel. Samples treated and untreated with LPS (+LPS and −LPS, respectively) were collected after 2 h incubation with LPS, and +LPS and −LPS samples incubated with peptide fraction or respective negative control were collected after 3 h (5 h since the beginning of the experiment) by cell detachment with Tryple Express (ThermoFisher Scientific), centrifugation at 500× g and pellet resuspension in NZYol (NZYtech, Lisbon, Portugal). RNA was extracted according to manufacturer’s instructions, cDNA synthesized using the NZY M-MULV First strand cDNA synthesis kit (NZYtech), and TNF-α and RNA 18S genes were amplified using the NZY Supreme qPCR Green Master Mix (NZYtech) in a Corbett-Rotor Gene thermal cycler (Qiagen, Hilden, Germany). The expression of TNF-α in samples was determined with the 2^−∆∆Ct method [79 (link)].