*5.6. Neutral Red Viability Assay*

The neutral red uptake assay was used to analyze the HGEC cell viability as previously described [28]. In summary, HGEC cells were grown with a complete medium, until confluence, in 96-well plates. After 72 h of treatments, freshly diluted neutral red (Sigma Aldrich, St. Louis, MO, USA) was added to cells to a final concentration of 10 mg/mL, followed by an additional incubation of 1 h at 37 ◦C in 5% CO2. Then, cells were washed and fixed with 1% CaCl2/1% formaldehyde, followed by lysis with 1% acetic acid in 50% ethanol. Absorbance at 540 nm was measured in an automated plate spectrophotometer. Results were expressed as viability percentage, in which 100% represents cells incubated under identical conditions but without treatment. The 100% of HGEC viability protection was considered when Stx2 cytotoxic effects were totally reversed.
