MCF 10A-H2B-mCherry cells were plated at densities that ranged from 156 to 5000 cells per well in 384-well plates using the Multidrop Combi Reagent Dispenser (Thermo Scientific) and grown for 24 hours. Cells were treated with a dilution series of drugs using a D300 Digital Dispenser (Hewlett-Packard) and imaged after drug addition in an Operetta (Perkin Elmer) for high content imaging system equipped with a live-cell chamber over a period of 72 hours.
In the case of methotrexate and oligomycin, 1250 cells were plated in 20–120 µl of media per well, treated with a dilution series of drug, and imaged for 72 hours.
In the case of linsitinib, cells were treated with a dilution series of linsitinib either with or without 10µM batimastat using a D300 Digital Dispenser and imaged in an IncuCyte ZOOM live cell imager (Essen Bioscience) for an additional 72 hours.
In the case of paclitaxel, cells were treated with a dilution series of paclitaxel and 200 nM of NucView 488 caspase 3 substrate (Biotium) using a D300 Digital Dispenser (Hewlett-Packard) and imaged after drug in an IncuCyte ZOOM live cell imager (Essen Bioscience) for an additional 72 hours. For immunofluorescence experiments, cells were grown for 24 hours and then treated with a dilution series of paclitaxel using a D300 Digital Dispenser (Hewlett-Packard) and incubated for 3, 6, 12, and 24 hours. Cells were fixed for 30 min in 3% formaldehyde, permeabilized for 30 min in phosphate buffered saline (PBS) with 0.3% Triton X-100 (Sigma-Aldrich), washed twice in PBS with 0.1% Tween 20 (Sigma-Aldrich; PBS-T), and blocked for 60 min with Odyssey blocking buffer. Anti-active Caspase-3 antibody (BD Biosciences) was diluted 1:1000 in Odyssey blocking buffer and incubated for 16 h at 4°C. Cells were washed three times in PBS-T for 5 min and incubated with Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody for 60 min at room. Cells were washed two times in PBS-T, once with PBS, and stained for 30 min with whole cell stain (Thermo Fisher Scientific) and Hoechst (Thermo Fisher Scientific), and washed three times in PBS.
In the case of methotrexate and oligomycin, 1250 cells were plated in 20–120 µl of media per well, treated with a dilution series of drug, and imaged for 72 hours.
In the case of linsitinib, cells were treated with a dilution series of linsitinib either with or without 10µM batimastat using a D300 Digital Dispenser and imaged in an IncuCyte ZOOM live cell imager (Essen Bioscience) for an additional 72 hours.
In the case of paclitaxel, cells were treated with a dilution series of paclitaxel and 200 nM of NucView 488 caspase 3 substrate (Biotium) using a D300 Digital Dispenser (Hewlett-Packard) and imaged after drug in an IncuCyte ZOOM live cell imager (Essen Bioscience) for an additional 72 hours. For immunofluorescence experiments, cells were grown for 24 hours and then treated with a dilution series of paclitaxel using a D300 Digital Dispenser (Hewlett-Packard) and incubated for 3, 6, 12, and 24 hours. Cells were fixed for 30 min in 3% formaldehyde, permeabilized for 30 min in phosphate buffered saline (PBS) with 0.3% Triton X-100 (Sigma-Aldrich), washed twice in PBS with 0.1% Tween 20 (Sigma-Aldrich; PBS-T), and blocked for 60 min with Odyssey blocking buffer. Anti-active Caspase-3 antibody (BD Biosciences) was diluted 1:1000 in Odyssey blocking buffer and incubated for 16 h at 4°C. Cells were washed three times in PBS-T for 5 min and incubated with Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody for 60 min at room. Cells were washed two times in PBS-T, once with PBS, and stained for 30 min with whole cell stain (Thermo Fisher Scientific) and Hoechst (Thermo Fisher Scientific), and washed three times in PBS.
