Cytotoxicity detection kit

STEC were cultured as a monolayer on a 12-well plate (Corning, I0905-191). For infection, cells were first washed with serum-free DMEM and incubated with bacteria in serum-free DMEM at 37°C. Cell death was examined by measuring lactate dehydrogenase release using a Cytotoxicity Detection kit (Beyotime, C0017). For autophagy analysis, a medium containing 10 μM CCCP for 6 and 12 h, 5 mM NAC for 1 h, 3 mM 3-MA for 3 h, or 1 μM Baf-A1 for 4 h.

Commercial cell counting Kit-8 (CCK-8) (Do-jindo, Kumamoto, Japan) was used to detect cell viability (Ishiyama et al., 1997 (link)). Primary astrocytes cultured to the third generation were seeded in 96-well plates at a density of 10⁴/well. The 96-well plates were placed in a cell culture incubator for 24 h before being subjected to OGD/R. Thereafter, 10 μL CCK-8 reagent was added to each well. The 96-well plates were then placed in the cell culture incubator for 2 h, and the absorbance at 450 nm was measured by a microplate reader (Multiskan, Thermo Scientific, Waltham, MA, United States).
Cytotoxicity was determined by measuring the lactate dehydrogenase (LDH) of the cell culture supernatant using the Cytotoxicity Detection Kit (C0016, Beyotime, Shanghai, China) according to the manufacturer’s instructions (Lobner, 2000 (link)). Briefly, the sample maximum enzyme activity control wells were set according to the instructions. Astrocytic supernatants from each group were centrifuged. In each well of the 96-well plate was added 120 μL supernatant and 60 μL reagent. Then, the 96-well plate was incubated at room temperature for 30 min in the dark, and the absorbance at 490 nm was measured by a microplate reader. Experiments were repeated five times, and each experiment contained five duplicate wells for each astrocyte group.

We ascertained cell viability by Cell Counting Kit-8 (CCK-8, CK04, Dojindo, Tokyo, Japan) assay. In brief, cells was cultured in a 96-well plate, and incubated with the reagent at 37 °C for 2 h. Then, optical density (OD) values were measured at 450 nm by a Thermo Multiskan FC microplate photometer.
Cellular injury-induced cytotoxicity was measured by Cytotoxicity Detection Kit (C0017, Beyotime Biotech, China) in line with the directions of the manufacturer.

The amount of LDH released by cells was detected using the Cytotoxicity Detection Kit (C0016, Beyotime). Cells were treated with 50, 100 or 200 μg/mL of SEP for 24 hours, centrifuged at 400 g for 5 minutes and the supernatant was removed. The LDH release reagent was diluted 1:10 in phosphate buffer saline (PBS) and then added to the pellet. One hour later, samples were centrifuged and 120 μL supernatant per well was transferred to a new plate and the plate was read on a plate reader (Thermo Fisher Scientific, Waltham, MA, USA) at an absorbance of 490 nm.

The ARPE-19 (human retinal pigment epithelial) cell line was purchased from ATCC (CRL2302) and cultured in a DMEM-F12 medium supplemented with 10% fetal bovine serum, 0.348% sodium bicarbonate, 2 mM L-glutamine, 100 μg/mL of streptomycin, and 100 U/mL of penicillin. The cell culture was maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ [50 (link)]. ARPE-19 cells were used within 10 generations, and the medium was changed every two days. COSs and NCOSs were dissolved with PBS buffer, filtered through a sterile 0.22 μm filter, and diluted with complete culture medium to different concentrations for the cell experiments.
The ARPE-19 cells were seeded in 96-well plates at 5 × 10⁴ cells per well and incubated overnight. After incubation with different concentrations of COSs or NCOSs for 48 h, the cells were treated with 75 μM acrolein for 24 h. Cell viability was measured via MTT cell proliferation and a cytotoxicity detection kit (Beyotime). After 4 h of incubation with MTT, the solubilization buffer was added to each well and incubated at 37 °C overnight. The optical densities were read at 555 nm using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA).