Vero Cell Cytotoxicity Assay for Ricin

Vero cell cytotoxicity assays were performed as described [29] (link). In brief, Vero cells were trypsinized, adjusted to ∼5 ×10⁴ cells per mL and seeded (100 µl/well) into white bottom 96-well plates (Corning Life Sciences, Corning, NY), and allowed to adhere overnight. Vero cells were then treated with ricin (0.01 µg/mL; 154 pM), ricin:Ab mixtures, or medium alone (negative control) for 2 h at 37°C. Cells were washed to remove non-internalized toxin or ricin:Ab mixtures, and 100 µl of fresh medium was added to the wells. Fresh medium was allowed to incubate for 48 h and cell viability was measured using CellTiter-Glo (Promega, Madison, WI). All samples were performed in quadruplicate and 100% viability was defined as the average value obtained from wells in which cells were treated with medium only.