20 70 dichlorofluorescein diacetate

Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Bio Whitaker Europe (Lonza, Madrid, Spain). DHCA, DHFA, HHA, gentamicin, penicillin, streptomycin, o-phthaldialdehyde (OPT), GR, GSH and oxidised (GSSG) glutathione, nicotine adenine dinucleotide (reduced) (NADH), nicotine adenine dinucleotide phosphate reduced salt (NADPH), 20,70-dichlorofluorescein diacetate (DCFHDA), 2,4-dinitrophenylhydrazone (DNPH), β-mercaptoethanol, guanidine, dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), dimethylsulphoxide (DMSO), t-BOOH, and sodium dodecyl sulphate (SDS) were purchased from Sigma-Aldrich (Madrid, Spain). Bradford reagent was acquired from Bio-Rad (Madrid, Spain). Recombinant murine TNF-α was from PreproTech (Tebu-bio, Madrid, Spain). IL-6 ELISA kit was from R&D Systems (Materlab, Madrid, Spain). The Pro Human Cytokine, Chemokine, and Growth Factor Assay (group I) Multiplex kit was purchased from Bio-Rad (Madrid, Spain). Cell culture dishes were from Falcon (Cajal, Madrid, Spain). Human hepatoma HepG2 cells were a kind gift from Dr. Paloma Martin-Sanz (Instituto de Investigaciones Biomédicas Alberto Sols, CSIC, Madrid, Spain). All other chemicals were of analytical grade.

MPO catalyzes the production of hypochlorous acid, which reacts with taurine to form taurine chloramine. TNB reacts with taurine chloramine to form a colorless product (DTNB). Therefore, the decrease in color intensity is an indication of higher MPO activity and concentration. Regarding the detection of caspase-1 activity, we applied a colorimetric technique that is based on the ability of caspase-1 to cleave p-nitroanilide to yellow product and detected at 405 nm. For the determination of MDA, incubation of tissue homogenate and thiobarbituric acid for 30 min at 95°C in acidic media will result in a thiobarbituric reactive product with pink color that is measured spectrophotometrically at 534 nm. GSH, and SOD were determined according to the manufacturer’s instructions. For the determination of ROS, briefly, distal colon tissue (200 mg) was homogenized in ice-cold Tris-HCl buffer (40 mM, pH = 7.4) (1:10 w/v). Then, homogenates (100 µl) were mixed with Tris-HCl buffer (1 ml), and 5 µl of 20, 70-dichlorofluorescein diacetate (10 µM) (Sigma-Aldrich) was added and the reactions were incubated for 30 min at 37°C. The fluorescence intensity (FI) was measured with a SpectraFluor Plus Microplate Reader Tecan, Mainz, Germany), λ excitation = 485 nm and λ emission = 525 nm.

According to Gupta et al. [26 (link)] methods, the reactive oxygen species formation (ROS) in liver tissue was described with some modifications. Liver samples (200 mg) were homogenized in ice-cold Tris-HCl buffer (40 mM, pH 7.4) (1:10 w/v). Then, 100 mL of samples of tissue homogenate were mixed with Tris-HCl buffer (1 mL) and 5 mL of 20,70-dichlorofluorescein diacetate (10 mM) (Sigma-Aldrich, St. Louis, MO). The mixture then was incubated for 30 min in 37°C. After the incubation, the fluorescence intensity of the samples was assessed using a FLUOstar Omega multifunctional microplate reader (λ_excitation 485 nm and λ_excitation 535 nm).