Mtt cell proliferation and cytotoxicity kit

2FF was provided by SynChem. Inc. (IL, United States). NAC was purchased from Sangon Biotech (Shanghai) Co., LTD. (Shanghai, China). Acetaminophen was provided by Abcam (CA, United States). The aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutathione (GSH) and malondialdehyde (MDA) test kits were provided by Jiancheng Bioengineering Institute (Nanjing, China). The lactate dehydrogenase (LDH) kit, MTT cell proliferation and cytotoxicity kit and SOD activity kit were provided by Beyotime Biotechnology (Nantong, China). Reactive Oxygen Species (ROS) Fluorescent Probe-Dihydroethidium (DHE) was provided by Vigorousbio. CO, Ltd. (Beijing, China). The mouse TNF-α and IL-6 ELISA kits were obtained from ABclonal Technology Co., Ltd. (Wuhan, China). Aleuria aurantia lectin (AAL), wheat germ agglutinin (WGA) and concanavalin A (ConA) were provided by Vectorlabs, Inc. (CA, United States). Antibodies against Nrf2, NQO1, keap1, NF-κB p65 and iNOS were from Abcam (Cambridge, United Kingdom). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), COX-2, HO-1 and Histone were from Proteintech (Wuhan, China).

2FF was provided by SynChem. Inc., Elk Grove Village, IL, USA. Hydrogen peroxide was purchased from Sangon Biotech (Shanghai, China). Catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) test kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The mitochondrial membrane potential (MMP) assay kit (JC-1 Kit), lactate dehydrogenase (LDH) kit, MTT cell proliferation and cytotoxicity kit and Mn-SOD activity kit were obtained from Beyotime Biotechnology, China. ROS Fluorescent Probe-Dihydroethidium (DHE) was purchased from Vigorousbio. CO., Ltd. (Beijing, China). Biotinylated aleuria aurantia lectin (AAL, B-1395, 1:3000) was purchased from Vectorlabs, Inc. (Burlingame, CA, USA). Antibodies against Nrf2 (ab62352, 1:1000), NQO1 (ab34173, 1:1000), keap1 (ab139729, 1:2000), NF-κB p65 (ab32536, 1:2000), NF-κB p65 (phospho S536, ab76302, 1:1000) and Nrf2 (phospho S40, ab76026, 1:2000) were from Abcam (Cambridge, UK). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60004, 1:5000), HO-1 (10701, 1:2000), cyclooxygenase-2 (COX-2, 66351, 1:1000), IκBα (10268, 1:1000) and Histone (10856, 1:1000) were from Proteintech (Wuhan, China).

The 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation and cytotoxicity kit (Beyotime, China) was used following the manufacturer. Briefly, cells of the B16–F10 melanoma line (CRL-6475, ATCC, China) were cultured in Dulbecco's Modified Eagle Media (DMEM) high-glucose medium (Gibco-Sigma, USA) in 96-well plates (5 × 10³ cells/100 μL medium) for 24 h. B16–F10 cells were treated with either 8-methoxypsoralen (0.05, 0.1, 0.15, 0.3, 0.5, and 1.0 mmol/L) for 24 h or PETM (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL) for 24 h or 48 h. 10 μL MTT (5 mg/mL) was added prior to another 4-h incubation. Formazan solubilization solution (100 μl) was then added to each well and mixed until the purple crystals were fully dissolved. Absorbance at 570 nm was measured by a microplate reader (Tecan, China). Percentages of cell viability was calculated as OD_Sample/OD_Blank × 100. Three independent experiments were performed with six technical replicates per experiment.