Multiplex Inflammation Profiling in Resistant Cells

For the simultaneous detection of 40 Human inflammatory factor concentrations in melatonin-treated HepG2/dox-resistant cell lysates, human inflammation antibody array- membrane (cat# ab 134,003, Abcam, USA) was used (Lu et al. 2020 (link)). The manufacturer protocol was followed. Briefly, after blocking the membrane array with 1X blocking buffer, the membrane was incubated with the sample lysate (140 µg) overnight at 4 °C. The membrane array was washed thoroughly with wash buffers I, and II that were supplied in the kit. The membrane was then incubated with 1 × Biotin-conjugated anti-cytokines overnight at 4 °C. After washing steps, the membrane was incubated with 1 × HRP (horseradish peroxidase)-conjugated streptavidin for 2 h at RT. The Chemiluminescence signals were detected by a CCD (charged-coupled device) camera of a chemiluminescence imager (UVP, UK) to digitally visualize protein spots on the developed membranes. Spot densitometric analysis of the arrays was performed using Visionworks ls (Analytik Jena, Germany). Then, the background signals were subtracted, and normalization to the positive control was calculated before comparing analyte-by-analyte to determine relative differences in cytokine expression in each sample.