4.2. LDH Assay

A lactate dehydrogenase (LDH) test was performed according to the manufacturer’s instructions (04744926001, Roche, Penzberg, Germany) to test for possible cytotoxic effects of leptin. For this purpose, the same amount of cell culture supernatant was mixed with LDH solution consisting of catalyst and staining solution in a ratio of 1:45 in a 96-well plate (10-121-0000, nerbe plus, Winsen/Luhe, Germany) and incubated for 30 min in the dark at room temperature. After addition of stop solution, the absorbance at 490 nm and 690 nm was measured with an ELISA reader (Multiscan GO, Thermo Fisher Scientific, Waltham, MA, USA). Background absorbances at 690 nm were subtracted from the primary wavelength measurement at 490 nm.