Amresco (Solon, OH, USA). Lipofermata was bought by Cayman Chemical Co. (Ann Arbor, MI, USA). The Pierce bicinchoninic acid protein assay kit was provided by Sigma-Aldrich (St. Louis, MO, USA). Nitrocellulose membranes and enhanced chemiluminescence reagents have been supplied by GE Healthcare Bio-Sciences (Pittsburgh, PA, USA) and Thermo Fisher Scientific (Pittsburgh, PA, USA), respectively. 2.2. Cell Culture and Remedies Human liver HepG2 cells (America Kind Culture Collection, Rockville, MD, USA) had been maintained in Dulbecco’s modified Eagle’s medium supplemented with 10 fetal bovine serum and 1 penicillin/streptomycin in CO2 incubator containing 5 CO2 at 37 C. The culture medium was changed each and every two days, and also the cells were sub-cultured into 96-well plates, 6-well plates, and T-25 flasks (SPL Life Sciences Co., Ltd., Pocheon, Korea) once they reached 800 confluency making use of 0.05 trypsin and 0.53 mM ethylenediaminetetraacetic acid. For the experiment, TEB was dissolved in DMSO at a concentration of 40 mM. The cells had been treated with 020 TEB or vehicle (DMSO) handle for 14 h. The concentration range of TEB for cell remedy was selected from prior studies [17,18].Foods 2021, ten,3 of2.3. Cell Viability Assay An MTT assay was performed to evaluate cell viability working with 3-(4,5-dimethylthiazol2yl)-2,BChE Formulation 5-diphenyl-2H-tetrazolium bromide (MTT; Amresco, Solon, OH, USA), as described previously [19]. HepG2 had been cultured into a 96-well plate and exposed to numerous concentrations of TEB (10, 20, 40, 80, 160, and 320 ) or DMSO for 24 h (n = three wells/group). The media have been replaced with 100 of fresh media and had been added with 10 MTT resolution (5 mg/mL in PBS). Subsequently, the cells had been incubated within a CO2 incubator containing 5 CO2 at 37 C for 3 h. following discarding 90 MTT resolution, 180 acidic isopropanol was added into each and every well, along with the plate was placed at 37 C for 1 h. The optical density (OD) was measured at 570 nm and 630 nm applying an Epoch spectrophotometer (BioTek Instruments, Winooski, VT, USA), and the percentage of cell viability was calculated together with the following Formula (1): Cell viability ( ) = (OD sample /OD control ) one hundred two.four. Lactate Dehydrogenase (LDH) Activity Assay An LDH release assay was carried out to evaluate the cell membrane integrity using the Cytox 96non-radioactive cytotoxicity assay kit (Promega, Madison, WI, USA). HepG2 cells had been cultured in a 96-well plate and exposed to several concentrations of TEB (10, 20, 40, 80, 160, and 320 ) or DMSO for 24 h (n = three wells/group). To measure the CA Ⅱ Molecular Weight maximum LDH release, 10lysis option was supplemented to the wells about 45 min prior to the end with the TEB remedy. Then, 50 of supernatants have been transferred into a brand new 96-well plate. The 96-well plate was incubated at 25 C for 30 min immediately after adding the CytoTox 96reagent of 50 to every nicely. The 50 of quit solution was then added to each and every nicely. The OD was measured at 490 nm employing a spectrophotometer, along with the percentage of LDH release was calculated with all the following Formula (two): LDH release ( ) = (OD Experimental LDH release)/(OD Maximum LDH release) 100 (two) 2.five. Oil Red O Staining Intracellular triglycerides and cholesterol esters have been determined employing Oil Red O (Sigma-Aldrich, St. Louis, MO, USA). HepG2 cells have been grown in 6-well plates and treated with 20, 40, and 80 TEB or DMSO for 24 h with or devoid of NAC pretreatment (five mM in PBS) for 1 h (n = three wells/group). The cells were fixed with ten formalin at 25 C for 1 h
