P nitrophenyl d glucuronide

LDH activity was determined after 16 h test material incubation using a Roche Cytotoxicity Kit (Sigma-Aldrich) as described by the manufacturer. The induced color change was measured photometrically with an Infinite F200Pro plate reader (Tecan Deutschland GmbH, Crailsheim, Germany). To measure GLU activity, 50 µL of the supernatant (sampled after 16 h incubation) was incubated under standard cell culture conditions with 100 µL 0.2 M sodium acetate buffer (pH 5) containing 13.3 mM p-nitrophenyl-d-glucuronide (Sigma-Aldrich) and 0.1% Triton X-100. The reaction was terminated after 2 h by addition of 100 µL 0.2 M sodium hydroxide. Optical density was measured with a plate reader at 405 nm. Both LDH- and GLU-based values were corrected for cell-free adsorption and normalized to the results of the positive control (0.1% Triton X-100 in F-12K assay medium).

To measure the amount of LDH and GLU released from the treated NR8383 cells, cell culture supernatants were harvested and centrifuged (10 min, 200 g) to remove cell debris. From each well, 50 μL was incubated with LDH reaction mix (Roche Cytotoxicity Kit; Roche, Germany) and evaluated as described by the manufacturer. Measurements were corrected for cell free-adsorption and normalized to the PC value (set to 100 %) obtained from NR8383 cells lysed with 0.1 % Triton X-100 (Sigma Aldrich, Germany). To measure GLU activity, 50 µL of the supernatant was incubated at 37 °C with 100 μL 0.2 M sodium acetate buffer (pH 5) containing 13.3 mM p-nitrophenyl-d-glucuronide (Sigma Aldrich, Germany) and 0.1 % Triton X-100 [50 (link)]. The reaction was terminated after 2 h by addition of 100 μL 0.2 M NaOH (Merck KGaA, Germany). Optical density (OD) was measured at 405 nm in a plate photometer (Tecan 200Pro, Tecan, Germany); values were corrected for cell free-adsorption and normalized to the PC value (set to 100 %) again obtained from NR8383 cells lysed with 0.1 % Triton X-100.