Enspire plate reader

The HER2-positive breast cancer cell line BT-474, SK-BR-3, gastric cancer cell line NCI-N87, ovarian cancer cell line SK-OV-3, weakly positive breast cancer cell line MCF-7 and HER2-negative breast cancer cell line MDA-MB-468 were obtained from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium (Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and Glutamax (Invitrogen). Cells (3.3 × 10⁴ cells/mL) were added to each well of a 384-well plate after which 10 μL compound was also added to the assay plate. The plate was incubated for 4 days at 37 °C, 5% CO₂, 95% humidity. Then the plates were incubated at room temperature for about 10 min, 40 μL CTG reagent was added to each well, and the plates were incubated for 30 min at room temperature. Luminescence was detected using the EnSpire Plate Reader, and Prism5 for Windows (Graphpad software, Inc., La Jolla, CA, USA) was used for data analysis, including IC₅₀ calculations.

Human HER2-positive cell lines (BT-474, HCC1954, and NCI-N87) and HER2-negative cell lines (MCF-7 and MDA-MB-468) were purchased from the ATCC. Tumor cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and GlutaMAX (Invitrogen, Carlsbad, CA, USA). Cells (3.3 × 10⁴ cells/well) were added to each well of 384-well plates and incubated at 37 °C overnight, after which 10 μL of compound aliquots were added to the assay plate. The plate was incubated for 7 days at 37 °C, 5% CO₂, and 95% humidity. Then, the plates were removed from the incubator and equilibrated at room temperature for 10 min. The Cell Titer Glo^® (CTG, Promega, Madison, WI, USA) reagents were incubated at 37 °C before the experiment. The buffer was equilibrated to room temperature and used to dissolve the substrate. Then, 40 μL of CTG reagent was added to each well for detection (at 1:1 ratio to the culture medium). Luminescence was detected using an EnSpire plate reader, and Prism5 for Windows (GraphPad software, Inc., La Jolla, CA, USA) was used for data analysis, including the 50% inhibition concentration (IC₅₀) calculations.