Ab65393

Measurement of LDH leakage was performed using a commercial LDH assay kit (ab65393, Abcam, USA). Briefly, 100-μL cells were seeded in a 96-well plate in triplicate. After incubation at 37 °C, the cells were centrifuged at 4 °C for 10 min, and the supernatant was transferred into a new 96-well plate. In each well, 100-μL LDH reaction mix was added, and the plate was incubated for 30 min at room temperature. The results were obtained using a microplate reader (Thermo Fisher Scientific, USA) with excitation at 450 nm and emission at 650 nm.

The Caco-2 cell line (human epithelial colorectal adenocarcinoma cell line; ATCC HTB-37) was seeded into a 24-well plate at a density of 1×10⁵ cells/well with a minimum essential medium (MEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin and incubated at 37°C and 5% CO₂ for 2 days. AO356 was treated on a Caco-2 cell with 1×10⁷, 1×10⁸, and 1×10⁹ CFU/well and incubated at 37°C and 5% CO₂ for 24 h. Escherichia coli (ACTC 1682) was used as a positive control. The test wa perfomed in triplicate. The supernatant was centrifuged at 5,000 g for 15 min and used as a sample. The toxicity of AO356 was determined using an LDH assay kit following the manufacturer’s protocol (ab65393, Abcam, Cambridge, UK). The non-treated culture medium was used as a low control, the cell lysis buffer was used as a high control, and Escherichia coli (ACTC1682), known to be toxic, was used as a positive control.

The production of active extracellular LDH in response to pesticide exposures was measured using an LDH assay kit (ab65393, Abcam, Cambridge, UK) according to the manufacturer’s instructions. After agent treatment (1–200 µM, or vehicle control), 50 µL of spent media was removed and LDH activity was measured spectrophotometrically at 450 nm (Multiskan Spectrum, Thermo Electron Corporation, Finland). Assays were performed in triplicates, with blank values from a negative control subtracted from test values. IC₅₀ values were obtained from the concentration-response curves and expressed as means ± standard error of the mean (SEM). Experiments were performed with an n-number of at least five.