Corresponding isotype control antibodies

To assess HER2 expression levels, cells were incubated either with phycoerythrin (PE)-labeled anti-human HER2 (24D2) or with corresponding isotype-control antibodies (BioLegend, San Diego, CA, USA) in buffer (1 % FBS, 2 mM EDTA and 0.1 % NaN₃ in PBS) for 30 min at 4 °C, after which they were washed and then analyzed using a MACSQuant Analyzer (Miltenyi Biotech K.K., Bergisch Gladbach, Germany). Before using the analyzer, 4 μg/ml propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, USA) was added to each sample to exclude dead cells.
To assess T-DM1 binding to PDA cells, 0.5x10⁶ cells were incubated with 30 μg/ml T-DM1 at 37 °C for 1 h. The cells were washed, incubated with PE-labeled anti-human IgG Fc (HP6017) (Biolegend) or corresponding isotype-control antibodies (Affymetrix, Santa Clara, CA, USA) in buffer for 30 min at 4 °C, washed, re-suspended and analyzed using a MACSQuant Analyzer (Miltenyi Biotech K.K.). Before using the analyzer, 4 μg/ml PI (Sigma-Aldrich) was added to the sample to exclude dead cells. The mean fluorescence intensity (MFI) of HER2 was analyzed using MACSQuantify Software.

MIA PaCa-2 cells were suspended in Hoechst 33342 (5 μg/ml) (Life Technologies) and incubated at 37 °C for 90 min, then washed and incubated with phycoerythrin (PE)-labeled anti-human HER2 (24D2) or the corresponding isotype control antibodies (BioLegend) for 30 min at 4 °C. Before using the analyzer, 1 μg/ml PI (Sigma-Aldrich) was added to the sample to exclude dead cells. In cell cycle analysis, the mean fluorescence intensity (MFI) of HER2 in each phase of the cell cycle was examined using MACSQuant Analyzer (Miltenyi Biotech K.K.) and MACSQuantify Software.