PBMC- and T cell-dependent cytotoxic activity of DCH and variant proteins. A two-color FACS®-based assay was performed. Kato III target cells prelabeled with the orange fluorescent dye PKH26 were incubated in a 16-h assay with immune effector cells (PBMCs or isolated human T cells) and DCH, variant proteins, or free cytokines at 37°C and 5% CO₂. A control reaction without fusion protein was included. The DNA-binding, red fluorescent dye PI was added at the end of the incubation, and cells were analyzed by flow cytometry using 585 nm (orange) and 660 nm (red) band pass filters. (A) PBMC-dependent cytotoxicity of 10 µg/ml DCH, variant proteins, and parental HMB without cytokines at an E/T ratio of 20:1. Recombinant human GM-CSF and IL-2, as well as the combination of both cytokines, were included at equimolar concentrations of 1.4 µg/ml. (B) T-cell dependent cytotoxicity of 10 µg/ml DCH, variant proteins, and parental HMB without cytokines at an E/T ratio of 10:1. Recombinant human GM-CSF and IL-2, as well as the combination of both cytokines, were included at equimolar concentrations of 1.4 µg/ml. Percentage of cytotoxicity (CT) was calculated as CT = [number of dead target cells (orange/red events) in sample / total number of target cells (orange events) in control] x 100. Error bars indicate standard deviations from duplicate determinations.