T-DM1 internalization assay

Zenon™ pHrodo™ iFL Red Human IgG Labeling Reagent ({"type":"entrez-nucleotide","attrs":{"text":"Z25612","term_id":"397198","term_text":"Z25612"}}Z25612; Thermo Fisher Scientific) was incubated with T-DM1 following manufacturer recommendations. We used a Molecular Probes® Cell Imaging Kit ({"type":"entrez-nucleotide","attrs":{"text":"R10477","term_id":"762433","term_text":"R10477"}}R10477; Thermo Fisher Scientific) containing NucGreen™ reagent (dead cell indicator) and NucBlue™ reagent (a total cell indicator). We seeded BT-474, SK-BR-3, MDA-MB-361 and MCF-7 in 3D 96-well plates for 24 h. As a positive control, we also cultured BT-474 in 2D 4-well chamber slides for 24 h. We next treated the 2D and 3D cells with Zenon™ pHrodo™ labeled T-DM1 (3 μg/mL). Some of the wells were incubated for time periods of 24, 48, and 72 h. Human IgG isotype control (#31154; Thermo Fisher Scientific) (3 μg/mL) labeled with Zenon™ pHrodo™ was used as a negative control. At the end of each treatment period, one drop of NucGreen™ reagent and one drop of NucBlue™ reagent were added to each well and incubated for 60 min prior to imaging with a Zeiss Axio Imager M2 fluorescent microscope (Zeiss, Thornwood, NY). The microscope was equipped with a Photometrics CoolSnap ES2 cooled monochrome camera (Teledyne Photometrics, Tucson, AZ) and appropriate filters (Chroma Technology, Bellows Falls, VT) for separation of NucGreen™, NucBlue™ and Zenon™ pHrodo™ fluorescent signals. The excitation/emission filters for NucBlue™, NucGreen™ and Zenon™ pHrodo™ iFL were 350 ± 25 nm/460 ± 25 nm, 490 ± 10 nm/520 ± 10 nm and 580 ± 12 nm/625 ± 15 nm, respectively. Image analysis was performed with FIJI (ImageJ) software.