MOLM-13, 2000cell/well were plated at set initial densities in 96-well culture plate with white bottom in the appropriate growth media. 48hours later, the medium was replaced. The ADCs in 10 ul of culture medium inconcentrations from 10^-5M to 10^-13M were then pipetted to the cells. After 6 hours of incubation at 37°C and 5%carbon dioxide, the medium was changed and the antibody-drug conjugates or metabolites were added by pipette in 10 ul of culture medium in concentrations of 10^-5M to 10^-13M to the cells (triplicates) in 90 ul. After 96 h, the cell proliferation was detected using the MTT assay (ATCC, Manassas, Virginia, USA, catalogue No. 30-1010K). To this end, the MTT reagent was incubated with the cells for 4 h, followed by lysis of the cells overnight byaddition of the detergent. The dye formed was detected at 570 nm (Infinite M1000 proTecan). The measured data were used to calculate the ICso of the growth inhibition usingthe DRC (dose response curve). The proliferation of cells which were not treated with testsubstance but were otherwise identically treated was defined as the 100% figure.

MV-4-11, 5000cell/well were plated at set initial densities in 96-well culture plate with white bottom in the appropriate growth media. 48hours later, the medium was replaced. The ADCs in 10 ul of culture medium inconcentrations from 10^-5M to 10^-13M were then pipetted to the cells. After 6 hours of incubation at 37°C and 5%carbon dioxide, the medium was changed and the antibody-drug conjugates or metabolites were added by pipette in 10 ul of culture medium in concentrations of 10^-5M to 10^-13M to the cells (triplicates) in 90 ul. After 96 h, the cell proliferation was detected using the MTT assay (ATCC, Manassas, Virginia, USA, catalogue No. 30-1010K). To this end, the MTT reagent was incubated with the cells for 4 h, followed by lysis of the cells overnight byaddition of the detergent. The dye formed was detected at 570 nm (Infinite M1000 proTecan). The measured data were used to calculate the ICso of the growth inhibition usingthe DRC (dose response curve). The proliferation of cells which were not treated with testsubstance but were otherwise identically treated was defined as the 100% figure.

MOLM-13, 2000cell/well were plated at set initial densities in 96-well culture plate with white bottom in the appropriate growth media. 48hours later, the medium was replaced. The ADCs in 10 ul of culture medium inconcentrations from 10^-5M to 10^-13M were then pipetted to the cells. After 6 hours of incubation at 37°C and 5%carbon dioxide, the medium was changed and the antibody-drug conjugates or metabolites were added by pipette in 10 ul of culture medium in concentrations of 10^-5M to 10^-13M to the cells (triplicates) in 90 ul. After 96 h, the cell proliferation was detected using the MTT assay (ATCC, Manassas, Virginia, USA, catalogue No. 30-1010K). To this end, the MTT reagent was incubated with the cells for 4 h, followed by lysis of the cells overnight byaddition of the detergent. The dye formed was detected at 570 nm (Infinite M1000 proTecan). The measured data were used to calculate the ICso of the growth inhibition usingthe DRC (dose response curve). The proliferation of cells which were not treated with testsubstance but were otherwise identically treated was defined as the 100% figure.

MV-4-11, 5000cell/well were plated at set initial densities in 96-well culture plate with white bottom in the appropriate growth media. 48hours later, the medium was replaced. The ADCs in 10 ul of culture medium inconcentrations from 10^-5M to 10^-13M were then pipetted to the cells. After 6 hours of incubation at 37°C and 5%carbon dioxide, the medium was changed and the antibody-drug conjugates or metabolites were added by pipette in 10 ul of culture medium in concentrations of 10^-5M to 10^-13M to the cells (triplicates) in 90 ul. After 96 h, the cell proliferation was detected using the MTT assay (ATCC, Manassas, Virginia, USA, catalogue No. 30-1010K). To this end, the MTT reagent was incubated with the cells for 4 h, followed by lysis of the cells overnight byaddition of the detergent. The dye formed was detected at 570 nm (Infinite M1000 proTecan). The measured data were used to calculate the ICso of the growth inhibition usingthe DRC (dose response curve). The proliferation of cells which were not treated with testsubstance but were otherwise identically treated was defined as the 100% figure.

After cell counting a defined cell number was sowed into a 96-well culture plate with white bottom (from Costar #3610) (MOLM13: 3000 cells/well) in total volume 100 uL. The cells (except MOLM-13 cells, these cells were directly treated) were then incubated in an incubator at 37°C and 5 % carbon dioxide. After 48 h, the medium was replaced. The treatment started by adding the test compounds in 10 uL of culture medium to the cells in triplicates. Concentrations ranging from 10^-5 M to 10^-13 M were chosen. Two identical treated sets of samples were prepared. One set was treated with the test compound alone and was added followed by a 96 h incubation in an incubator at 37°C and 5 % carbon dioxide.

After cell counting a defined cell number was sowed into a 96-well culture plate with white bottom (from Costar #3610) (MOLM13: 3000 cells/well) in total volume 100 uL. The cells (except MOLM-13 cells, these cells were directly treated) were then incubated in an incubator at 37°C and 5 % carbon dioxide. After 48 h, the medium was replaced. The treatment started by adding the test compounds in 10 uL of culture medium to the cells in triplicates. Concentrations ranging from 10^-5 M to 10^-13 M were chosen. Two identical treated sets of samples were prepared. One set was treated with the test compound alone, whereas to the second set the test compound and 5nM hum.neutrophil elastase were added followed by a 96 h incubation in an incubator at 37°C and 5 % carbon dioxide.