Ez read biochrom 400 microplate reader

The assay is based on the ability of living cells to reduce the MTS tetrazolium compound to a coloured formazan product soluble in the cell culture medium. The reduction is carried out by an NAD(P)H-dependent dehydrogenase in metabolically active cells. The HIEC-6 and the hepatocytes were placed onto 96-well-plates and incubated for 24 h with the inhibitors at 5, 20, 50, and 100 µM concentrations. The MTS assay was performed with eight parallels at each inhibitor concentration. After removing the medium and three times washing of the cells with PBS, 20 µL of CellTiter96 Aqueous One Solution (Promega Corporation, Madison, WI) containing MTS and an electron acceptor reagent, phenazine ethosulfate, were pipetted into a 96-well plate, each containing 100 µL of phenol red free medium. The plate was incubated for 1.5 h in a 5% CO₂ incubator. The viability of HIEC-6 was detected with an EZ Read Biochrom 400 microplate reader (Biochrom, Cambridge, UK) at 490 nm.

After 2 h inhibitor treatment (50 μM), HepK2 and HepK6 cocultures were incubated in the medium for 6 h. Culture media were collected, centrifuged (245 ×g for 10 min at room temperature), and diluted to measure the IL-6 and IL-8 levels.
Changes in IL-6 and IL-8 concentrations relative to those in controls were determined by porcine-specific IL-6 (Sigma-Aldrich, St. Louis, MO, USA) and IL-8 (Abcam, Cambridge, UK) ELISA kits according to the manufacturer's instructions. The absorbance values were detected at 450 nm using EZ Read Biochrom 400 microplate reader (Biochrom Ltd., UK).

tBP samples with a size of approximately 1 × 1 cm2 were weighed and degraded in 0.25 mg/mL collagenase at 37 °C. After 6, 24, and 48 h of incubation, the enzyme activity was stopped by adding 0.1 mL of EDTA solution (Sigma-Aldrich, USA). The digestion supernatants were collected and hydrolyzed with 37% H2SO4 at 120 °C for 3 h. The amount of hydroxyproline in the supernatant was determined by a hydroxyproline kit (Sigma-Aldrich, USA) using a colorimetric method at 570 nm. The final hydrolyzation samples were neutralized and added with chloramine-T in an oxidation buffer. The solutions were left to stand at room temperature for 5 min and subsequently added with DMAB/perchloric acid/isopropanol solution for the next step of chromophore development by incubation at 60 °C for 90 min according to the manufacturer’s protocol. This chromophore was measured with a Biochrom EZ Read 400 Microplate Reader (Biochrom, USA). The hydroxyproline content in the digestion supernatant was calculated using a calibration curve obtained with standard amounts of hydroxyproline. All degradation experiments were performed in triplicate.

All chemicals used in the cell culture experiments were purchased from Sigma-Aldrich (Darmstadt, Germany), including purified GSOP, LUT, dimethyl sulfoxide (DMSO), lipopolysaccharides (suitable for cell culture, derived from Salmonella enterica ser. Typhimurium, Escherichia coli O111:B4 and E. coli O127:B8), growth medium of IPEC-J2 cells, Neutral Red dye and 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA). Mueller-Hinton liquid broth was ordered from Biolab Ltd. (Budapest, Hungary). All cell culture plates were obtained from Corning Inc. (Corning, New York, NY, USA), while microplates used for minimum inhibitory concentration (MIC) determination were supplied by VWR International (Radnor, PA, USA). The Biochrom EZ Read 400 Microplate Reader is manufactured by Biochrom Ltd. (Cambridge, UK), while Victor X2 2030 fluorometer is the product of PerkinElmer Inc. (Waltham, MA, USA). R software is developed by R Foundation (Vienna, Austria).