LDH Release Assay

The MODE-K cell line was maintained in Dulbecco’s Modified Eagle Medium (DMEM; Sigma) supplemented with 10 % FBS at 37°C in 5 % CO₂. Bone-marrow-derived macrophages (BMDMs) were differentiated from bone marrow cells collected from femurs and tibias of SPF C57BL/6 mice. Briefly, bone marrow cells were harvested with 10 ml of cold RPMI-1640 medium (Sigma) and then pelleted at 1,000 rpm for 5 min at 25 °C. The cells were re-suspended in BMM medium (RPMI-1640 supplemented with 10 % heat-inactivated FBS, 1 mM glutamine, 1% antibiotics-antimycotics, and 30 % L-cell conditioned medium) and allowed for differentiation for 7 days. MODE-K experiments were performed in triplicate. Plates were seeded with cells to a final confluency of 80 % before treatment. Two days after seeding, MODE-K cells were treated for 24 h with 2–4 % DSS (Alfa Aesar), with and without 0.002–0.2 % sodium tungstate dihydrate (Sigma). For BMDM experiments, plates were seeded with 1×10⁵ cells per well for 48 h. After 24h the media was replaced to RPMI supplemented with 2 % FBS and glutamine. On the day of the experiment, culture media was replaced with media supplemented with 4 or 6 % DSS, with and without 0.2% sodium tungstate dehydrate, for 24 h. Cytotoxicity was determined using the LDH release assay CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega), per the manufacturer’s recommendations. Absorbance readings were corrected based on the absorbance of the medium alone. Five-minute treatment with 10 % Triton X-100 was used as the total LDH release control. The experiment was repeated 3 times and the representive results were shown.