Cytotoxicity assay kit

Lactate dehydrogenase (LDH) is a stable cytosolic enzyme released into the supernatant as a result of cell membrane damage and cell lysis. The increase of LDH activity in the supernatant was correlated with the percentage of necrotic cells. LDH activity measurement was performed using the cytotoxicity assay kit from Roche Diagnostics GmbH (Mannheim, Germany). The LNCaP cells (1 × 10⁶ cells/ mL) were treated with TRAIL (50–200 ng/mL) and/or xanthohumol (25 and 50 µM). Maximal LDH release was obtained after the treatment of control cells with 1% solution of Triton X-100 (Sigma Chemical Company) for 10 min at room temperature. The spectrophotometric absorbance was measured at 490 nm using the Eon microplate reader. The necrotic cell percentage was calculated using the formula: (sample value/maximal release) × 100% [53 (link),70 (link)].

IPA (Figure 1A) was provided by Dr. Young-Ho Kim (Chungnam National University, Daejeon, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, and trypsin were purchased from Welgene (Gyeongsan, Korea). STO-609, zinc protoporphyrin IX (ZnPP), ML385, and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compound C were obtained from Tocris (Cookson, Bristol, UK), and EDTA was purchased from GenDEPOT (Barker, TX, USA). W7 was obtained from Calbiochem (La Jolla, CA, USA). Antibodies against p-NF-κB p65, p-IκB, IκB, p-AMPK, p-CaMKKβ, p-GSK3β(Ser9), and p-LKB1 were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NF-κB p65, HO-1, NOQ1, Nrf2, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lamin B1 was purchased from Bioss Antibody, Inc. (Woburn, MA, USA). Tetrazole 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was acquired from USB Corporation (Cleveland, OH, USA). The cytotoxicity assay kit was obtained from Roche Applied Science (Indianapolis, IN, USA). All other chemicals were of the highest grade commercially available.

Mouse macrophage J774 was inoculated into 96-well plates at a density of approximately 20,000 cells per well. The cells were then cocultured with 200 μL of S. aureus at 37°C for 5 h. The cells were washed by PBS and stained with live/dead (green/red) reagent (Cat number 04511-1KT-F, Sigma-Aldrich), and the results were observed under a fluorescence microscope. The cytotoxicity assay kit (Cat number 11644793001, Roche, Basel, Switzerland) was used to assess the release of LDH from the supernatant in the wells.