Cytotoxicity Assay for THP-1 Cells

THP-1 cell cytotoxicity assays were done as described [40] (link). Briefly, THP-1 cells were spun (5 min at 400×g) and adjusted to ∼5 ×10⁴ cells per mL and seeded (100 µl/well) into clear U-bottom 96-well plates (BD Bioscience, San Jose CA) and allowed to grow overnight. The next day, THP-1 cells were spun to remove medium and were then treated with ricin (0.01 µg/mL; 154 pM), ricin:Ab mixtures, or medium alone for 2 h at 37°C. Cells were then subjected to centrifugation and washed to remove non-internalized toxin or ricin:Ab mixtures. Fresh medium was added to the wells and allowed to incubate for 48 h. Cell viability was determined using CellTiter-Glo after the content of each plate was transferred into white bottom 96-well plates. In order to address post-attachment experiments, THP-1 cells were kept on ice at 4°C, to allow for ricin attachment but prevented internalization of the toxin prior to antibody treatment. The cytotoxicity assay was performed as described above but cells were kept on ice until they were transferred to 37°C for the 48 h incubation period. 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.