Ldh diagnostic kit

The LDH activity was measured by using a LDH diagnostic kit (Stanbio Laboratory, USA) according to the manufacturer's protocol. Briefly, the PC12 cells were seeded on 24-well culture plates at a density of 1 × 10⁵ cells/well. At the end of the treatment, the medium was collected to measure the LDH activity. The absorbance of the samples was recorded at 340 nm at 1 min intervals for 3 min. The change in absorbance was then expressed in concentration units per liter. To determine the intracellular LDH activity, the cells were washed by D-Hanks and then 500 μL D-Hanks were added into each well. The cells were lysed by ultrasonication (750 W) for 30 s. The suspension was centrifuged and the supernatants were collected for assay of the LDH activity. The LDH leakage was expressed as the percentage (%) of the total LDH activity (LDH in the medium + LDH in the cell), according to the equation % LDH released = (LDH activity in the medium/total LDH activity) × 100.

Hydrogen peroxide (H₂O₂) (number 070610) was purchased from Nanjing Chemical Reagent Factory. RPMI-1640 medium, fetal bovine serum, 0.5% trypsin EDTA, penicillin, and streptomycin were purchased from Gibco (Grand Island, NY, USA). LDH diagnostic kit was purchased from Stanbio Laboratory, USA. Hoechst 33258, JC-1, fura-2/AM, ROS, and INOS were purchased from Beyontime. Propidium iodide (PI) was purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). The specific monoclonal anti-bcl-2 antibody, anti-bax antibody, and anti-caspase-3 antibody were obtained from Cell Signaling Int. All other chemicals and reagents were of analytical grade.