Ricin Cytotoxicity Assay Protocol

Ricin toxin (Ricinus communis agglutinin II) was purchased from Vector Laboratories (Burlingame, CA). Ricin was dialyzed against phosphate-buffered saline (PBS) at 4°C in 10,000-molecular-weight (MW)-cutoff Slide-A-Lyzer dialysis cassettes (Pierce, Rockford, IL), prior to use in cytotoxicity studies. Fetal calf serum was purchased from Gibco-Invitrogen (Carlsbad, CA). Cells were maintained in a humidified incubator at 37°C with 5% CO₂. Recombinant human tumor necrosis factor alpha (TNF-α), recombinant human soluble TRAIL (sTRAIL)/Apo2L/CD253, anti-human sTRAIL-(s)-Apo2L, and rabbit polyclonal anti-TRAIL antibody were purchased from Peprotech (Rocky Hill, NJ). Human TNF-α neutralizing rabbit Ab was purchased from Cell Signaling Technology (Danvers, MA). Z-LEHD-FMK, Z-VAD-FMK, Z-DEVD-FMK, and Z-IETD-FMK were purchased from ApexbBio (Taiwan). Necrostatin-1 (Nec-1), GSK′872, and necrosulfonamide (NSA) were purchased from EMD Millipore (Burlington, MA). Unless noted otherwise, all other chemicals were obtained from MilliporeSigma (St. Louis, MO). Murine MAbs against ricin toxin’s A subunit (PB10, SyH7, GD12, and IB2) and B subunit (24B11, SylH3, MH3, 8A1, 8B3, LF1, and LC5) were purified by protein A chromatography at the Dana Farber Cancer Institute (DFCI) Monoclonal Antibody Core facility (Boston, MA), as described previously (36 (link), 55 (link)).