Ldh cytotoxicity kitplus

Cell membrane integrity was evaluated using the lactate dehydrogenase (LDH) Cytotoxicity Kit^PLUS (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, 50 μL of cell supernatants were incubated with 50 μL of assay substrate for 10–30 min, at room temperature, protected from light. Absorbance readings were measured at 490 nm, with 620 nm reference wavelengths using a Bio-Rad Model 680 microplate reader. Further, cell death was also determined using the ToxiLight^TM BioAssay Kit (Lonza Walkersville Inc., Walkersville, MD, USA), according to the manufacturer’s instructions. The release of adenylate kinase enzyme from damaged cells was determined using 10 µL each of cell supernatants and bioluminescent cytolysis assay in the microplate reader.

BV2 cells were plated in 96-well plates at 5 × 10³ cells/well and transfected the next day for 24 h before exposure to 100 ng/mL LPS for additional 24 h. SH-SY5Y cells, with or without overexpression of α-syn, were plated in 96-well plates at 4 × 10⁴ cells/well and transfected the next day for 24 h. Cellular metabolic activity was determined using CellTiter 96^® Aqueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega, Madison, WI, United States). Alterations in absorbance were measured at 490 nm using GloMax^® Multi Detection System (Sunnyvale, CA, United States). Cell membrane integrity was determined using the lactate dehydrogenase (LDH) Cytotoxicity Kit^PLUS (Roche Diagnostics GmbH, Mannheim, Germany). Absorbance was measured at 490 nm, with 620 nm reference wavelengths, using a BioRad Model 680 microplate reader.