Vero cytotoxicity test

The Vero cell line (African green monkey kidney cell; ATCC^® CCL81) was used to determine the cytotoxicity of toxins produced by E. coli. The Vero cells were cultured in Dulbecco’s modified Eagle medium (DMEM; GIBCO, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU penicillin/mL, and 100 g of streptomycin/mL in 25 cm² culture flask at 37°C with 5% CO₂ humidified incubator. Around 80%–90% of confluent cells were disaggregated by 1× trypsin–ethylenediaminetetraacetic acid and seeded at 1 × 10⁵ cells/well (100 μL) into 96-well plates. The plates were cultured at 37°C, 24 h, with 5% CO₂ humidified incubator. Next, 100 μL of cell-free supernatant was added to each well and incubated for 48 h. Cell morphology was observed under an inverted microscope to check the cytotoxic effect every day [16]. Cell viability was determined by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay [17]. The supernatant was removed by gently pipetting. After that, 10 μL of 0.5 mg/mL MTT was added and incubated for 2 h. The formazan crystal was dissolved by adding 200 μL of DMSO (dimethyl sulfoxide) and incubated for 1 h. The optical density (OD) was determined at 570 nm by using a microplate reader. The percentage of cell viability was calculated using the following formula.

Viability (%) = 100 × OD570_Sample/OD570_Control