Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.

Cells, plated in triplicates at 3 × 103 cells per well in 96-well plates, were dosed with eight series of fourfold serially diluted PNU conjugates, unconjugated FL1, and free toxin (Creative Biolabs, #ADC-P-025) in 100 ul of complete growth media. Control cells were incubated with PBS-supplemented media. Cell viability was measured after 3 or 5 days of incubation, using CellTiter 96 Aqueous one-solution cell-proliferation assay reagent (MTS, Promega, #G358), as per the vendor's recommendations. Plate absorbance was then measured at 490 and 650 nm (for background subtraction) using a Spectra Max Plus plate reader (Molecular Devices). Data were plotted as a function of drug concentration (nanomolar) in GraphPad Prism 9 and fitted to a four-parameter nonlinear regression function, from which EC50 values were derived.