Detection of Leakage Rate of Lactate Dehydrogenase (LDH)
LDH was determined by a CytoTox 96® Non-Radioactive Cytotoxicity assay
kit (Promega Corporation, Madison, WI, USA). Cells were inoculated into
a 96-well plate at a density of 105 - 106 and incubated with 5% CO2 at
37°C. After cells were treated with high glucose and free fatty acids
(HG-FFA) for 1 h, 15 μL of lysis buffer [9% (v/v) Triton X-100
dissolved in water] was added to each well, and cells were incubated
at 37°C for 45 min. Then 50 μL supernatant was transferred to another
96-well plate, which was then reacted with a pre-formulated substrate
mixture (50 μL) at room temperature for 30 min in dark. Finally, 50 μL
of stop solution was added to each well to terminate the enzyme
reaction. And the OD value was measured at 490 nm by enzyme labeling
instrument.