Lactate dehydrogenase ldh cytotoxicity assay kit 2

Mouse Sertoli TM4 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). MTS cell proliferation assay kit, lactate dehydrogenase (LDH)-cytotoxicity assay kit II, 2’7’-dichlorofluorescin diacetate (DCFDA) cellular reactive oxygen species (ROS) detection assay kit, lipid peroxidation assay kit (malondialdehyde [MDA]), and GR assay kit were purchased from Abcam (Cambridge, MA, USA). API was purchased from Sigma-Aldrich (St. Louis, MO, USA) and mixed with dimethyl sulfoxide solution (DMSO) before being filtered.

S. pyogenes strains in late-log phase (OD_{600 nm} = 0.4) were spun down (6000× g, 3 min, RT) and washed once with 1 mL of phosphate-buffered saline (PBS). Bacterial suspensions were passed 10 times through a 27 Gauge syringe insert (NeoLab, Heidelberg, Germany) in a 1 mL syringe (VWR) to separate the cocci chains, and 10⁶ BLaER1 cells were infected at MOI 1. Plates were centrifuged (300× g, 5 min, RT) to synchronize the infection. P/S was added after 1 h to a final concentration of 1% to inhibit bacterial replication and excessive cell death. After overnight incubation, plates were centrifuged (500× g, 5 min, RT), after which supernatants were collected and stored at −20 °C until use. Supernatants were thawed on ice and IL-6 and IL-1β concentrations were measured using Enzyme-Linked ImmunoSorbent Assay (ELISA) kits (Thermofisher Scientific, Darmstadt, Germany) according to the manufacturer’s protocol. ELISA data were analyzed by extrapolating values of each sample from the standard curve using a log-log fit. The standard curve was generated for each plate by plotting the logarithm of the absorbance of the standards against the logarithm of the concentrations of each standard. In contrast, fresh supernatants were used to measure cell death using the Lactate Dehydrogenase (LDH)-Cytotoxicity Assay Kit II (Abcam, Cambridge, UK) according to the manufacturer’s instructions.