Lactate dehydrogenase cytotoxicity detection kit

L6 myoblast cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Human primary skeletal muscle-derived cells (HSKMDC) (catalogue #SK-1111, Lot #P0100750F) were obtained from Cook Myosite Ltd. (Pittsburgh, PA, USA). All forms of Dulbecco’s Modified Eagle Medium (DMEM), rat tail collagen I, media supplements, and cell culture reagents were from Life Technologies (Paisley, UK). The lactate dehydrogenase cytotoxicity detection kit was from Roche Diagnostics Ltd. (West Sussex, UK). ATP lysis buffer and ATP reagent kit were from Sigma Aldrich (Southampton, UK). Extracellular flux analyser (XF^e96) consumables and base medium were from Agilent Technologies (Santa Clara, CA, USA). Simvastatin hydroxy acid ammonium salt was from Toronto Research Chemicals (LGC Promochem, Middlesex, UK), and the control drugs propofol and pregabalin were purchased from United States Pharmacopoeia (Staten Island, NY, USA) and Merck (West Point, PA, USA), respectively. All other drugs, reagents, and chemicals were purchased from Sigma Aldrich (Dorset, UK).

Two weeks after the final immunization, splenocytes cells were retrieved from immunized mice and co-cultured with inactivated CVB3 for 3 days and used as effector cells. The plasmid pcDNA3.1-vp1 stably transfected autologous SP2/0 cells were used as target cells. A nonradioactive cytotoxic T lymphocyte assay (CTL) was performed using a lactate dehydrogenase cytotoxicity detection kit (Roche) according to the instructions. Briefly, effector cells and target cells were titrated in U-bottom 96-well tissue culture plates at the E/T ratio as 50:1, 25:1, and 12.5:1, and then 1 × 10⁴ target cells were added. After incubating at 37 °C for 4 h, 50 μL of cell supernatant per well was removed and transferred into the corresponding wells of a 96-well plate. Reaction mixture (50 μL) was added to each well and incubated for 30 min at room temperature. Then absorbance of the samples at 450 nm was measured. The percentage cytotoxicity of CTL was calculated as follows: Cytotoxicity (%) = [(effector and target cell mix–effector cell control) – low control]/(high control–low control)] × 100%.

WY-14643, GW6471, fenofibrate, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were purchased from Sigma Chemicals Co. (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium, fetal bovine serum, and penicillin-streptomycin solution were purchased from Welgene (Gyeongsan, Republic of Korea). Lipofectamine™ 2000 transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA). Enhanced chemiluminescence solution was purchased from Biofact (Daejeon, Republic of Korea). Nitrocellulose membrane was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). A luciferase assay system was obtained from Promega (Madison, WI, USA). pCMV-β-galactosidase was purchased from TaKaRa Bio (Kusatsu, Japan). Oligonucleotide polymerase chain reaction (PCR) primers were custom synthesized by Bioneer (Seoul, Republic of Korea). Antibodies against β-actin, CYP1B1, and PPARα were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and horseradish peroxidase-linked anti-rabbit and anti-mouse secondary IgGs were purchased from Cell Signaling Technology (Beverly, MA, USA). A 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay kit was purchased from Abcam (Cambridge, MA, USA), and a lactate dehydrogenase cytotoxicity detection kit was purchased from Roche (Mannheim, Germany). All chemicals were of the highest commercially available grade.