Alanine transaminase activity assay kit

Blood was collected and stood for 2 h at room temperature, and at 4°C overnight. Subsequently, blood samples were centrifuged at 4,000 × g for 10 min at 4°C to isolate the serum samples. The serum contents of ALT and AST were detected by using Alanine Transaminase Activity Assay kit (Cat#: ab105134, Abcam) and Aspartate Aminotransferase Activity Assay kit (Cat#: ab105135, Abcam) according to the operation instruction.

ALT and AST activity were determined in EDTA-treated plasma using the Alanine Transaminase Activity Assay Kit (ab105134) and the Aspartate Aminotransferase Activity Assay Kit (ab138878) from Abcam. Both assays were performed following the manufacturer’s recommendations. Samples were run together, analysed in duplicate wells per mouse sample, and the mean value of these technical replicates was used for subsequent analysis.

ALT activity was determined in liver lysates from the WT and Ack1 KO mice using the Alanine Transaminase Activity Assay Kit (Abcam), according to the manufacturer’s recommendations. The experiment was done in triplicates and the mean value has been plotted.

Mice were fasted for 6 h and then they were administered a 33% (vol/vol) ethanol solution at a total cumulative dosage of 4.5-g/kg by four equally divided gavages in 20-minute intervals, as described in Ding et al.^{72 (link)}. Control mice received the same volume of water. Vehicle (DMSO) or DMC were intraperitoneally injected to mice 30 min before ethanol administration. Sub-mandibular blood collection occurred 16 h after ethanol binge. Hepatic damage was quantified as serum ALT activity (Alanine Aminotransferase activity) by means of a specific kit (Alanine Transaminase Activity Assay Kit, Abcam [ab105134]). ALT activity was calculated as [mU*mg⁻¹] of pyruvate produced in the reaction.

ALT, AST, urea, albumin, and SOD assays were performed to estimate the impact of plant extracts on the liver tumor MPS. Alanine Transaminase Activity Assay Kit (catalog # ab105134, Abcam, Waltham, MA, USA), Aspartate Aminotransferase Activity Assay Kit (catalog # ab105135, Abcam, Waltham, MA, USA), Urea Assay Kit (catalog # ab83362, Abcam, Waltham, MA, USA), Human Albumin ELISA Kit (catalog # ab179887, Abcam, Waltham, MA, USA), and Superoxide Dismutase Activity Assay Kit (catalog # ab65354, Abcam, Waltham, MA, USA) kits were used for ALT, AST, urea, albumin, and SOD quantification, respectively. Cell culture media samples were briefly taken at specific time intervals and immediately stored at −80 °C. In contrast, an SOD assay was performed on the cell lysate prepared by the procedure previously described. Before the biomarker estimation procedure, cell culture media samples were thawed in a water bath at 37 °C. A semi-automated microplate reader (SpectraMax iD3, Molecular Devices, Silicon Valley, CA, USA) was utilized to take the readings per the manufacturer’s instructions.