Assessing Cell Viability with Immunotoxins

Viability of cell lines treated with immunotoxins was measured using the Cell Counting Kit-8 WST-8 assay (Dojindo Molecular Technologies, Inc.). Cells (2000 cells/well) were plated in 96-well plates, left overnight to adhere, and incubated with varying concentrations of RITs for 72 h at a final volume of 0.2 mL. For assays using PPCI (EMD Millipore, Billerica, MA), PPCI was added to the culture media immediately prior to plating the cells and included with the RITs to maintain the appropriate concentration. At the end of the incubation period, 10 μL of the CCK-8 reagent was added to each well and the plates were incubated at 37 °C until the wells with the maximum absorbance at 450 nm reached values of ~1 OD. Values were normalized between controls of cycloheximide (10 μg/mL) and buffer (Dulbecco’s phosphate buffered saline without Ca and Mg (D-PBS) containing 0.2% human serum albumin), then fit to a sigmoidal equation with variable slopes for the plateau, baseline, and Hill slope using GraphPad PRISM software. The equation was subsequently used to interpolate the concentration of RIT, which reduced cell viability to the 50% level (EC₅₀). Cells from patients with HCL were similarly evaluated using a protein synthesis inhibition assay^{36 (link)} and an ATP depletion assay^{39 (link)} as previously described.