Measurement of Lactate Dehydrogenase Activity

At 6 h or 24 h after the initial treatment with triclocarban, lactate dehydrogenase (LDH) that was released from damaged cells into the cell culture media was measured. The LDH release was estimated as previously described [18, 37]. The supernatants were collected from each well and incubated at room temperature for 30 to 60 min with the relevant reagent mixture according to the supplier’s instructions (Cytotoxicity Detection Kit). The intensity of the red color formed in the assay, measured at a wavelength of 490 nm, was proportional to both LDH activity and the number of damaged cells. The data from three to four independent experiments were normalized to the color intensity of the vehicle-treated cells (100%) and expressed as a percentage of control.