Cellular membrane integrity: Lactate Dehydrogenase assay

The release of the intracellular enzyme lactate dehydrogenase (LDH), indicative of cell membrane damage, was assessed by a LDH kit (Abcam) according to the manufacturer’s guidelines. Briefly, after cell exposure to the NFC samples, cell culture supernatants were collected and adherent cells were lysated for 30 min with cell lysis buffer diluted in cell culture medium (1:10) and the lysates were collected. Lysates and supernatants were centrifuged at 6800 g during 10 min to avoid any potential interference of NFC. The enzyme activity in the lysates and supernatants samples was measured by reading the absorbance at 450 nm wavelength and reference wavelength 650 nm using a plate reader (Tecan Infinite M200). The experiments were conducted at least three times in duplicate wells for each dose. LDH release (LDH activity in cell culture supernatant) was normalized by the total LDH activity (sum of LDH activity in cell culture supernatants and lysates) which correlates with the total number of cells in order to avoid any underestimation of toxicity [22].