Celltiter glo 3d kit

For 2D adherent cell viability experiment, the cells were seeded at 384-well plate (Corning, no. 3570) at the density of 200 cells/well. The next day, compounds were added using Janus workstation (PerkinElmer). After 5 days treatment, the cell viability was measured by CellTiter-Glo kit (Promega, no. G7570) as the manufacturer recommended. For 3D spheroid assays, NCI-H226 and MSTO-211H cells were plated at the density of 200 cells/well in Ultra-low Attachment (ULA) plate (S-bio, no. MS-9384WZ) without or with 5% Matrigel matrix (Corning, no. 356231) respectively. The cell viability was measured using 3D CellTiter-Glo kit (Promega, no. G9681). The luminescent signal was collected on EnVision plate reader (PerkinElmer). The GR₅₀ values were calculated as previously described Hafner et al., 2016 (link).

MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were seeded in DMEM-10% FBS in a 96-well cell culture plate with 10,000 cells per well. After cell attachment, various concentrations of lapatinib (Selleckchem) and paclitaxel (Selleckchem) were used to treat the cells. Following 48-h treatments, MTS (Sigma) was added to the wells according to the manufacturer’s instructions to obtain dose–response curve. MC-BR-BTY-0019 and MC-BR-BTY-0006 organoids were grown from corresponding PDX tumors freshly harvested from mice and cultured in MEF media with 10,000 cells per well in nanoculture plates. Briefly, PDX tumors were collected when the tumor grew to ~10–20 mm diameter. Primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described^{7 (link)}. A single-cell suspension of primary breast cancer cells was initially cultured in MEF media (DMEM with 10% FBS supplemented with glutamax, MEM NEAA, sodium pyruvate, and 5 μM Y-27632 ROCK inhibitor (Tocris)). ROCK inhibitor was removed from media by changing media one week before drug treatment. Cells were treated with various concentrations of lapatinib or paclitaxel for 5 days. 3D cell TiterGlo kit (Promega) was used to measure 3D culture viability according to the manufacturer’s instructions to obtain dose–response curves.

Cells were plated on 96-well plates at a density of 10,000 (SK-N-BE(2)-C, IMR-32, Kelly) or 20,000 (NB8) cells per well and treated as indicated for 72 h. According to manufacturer’s protocol, cells were incubated with reagent for 25 min using the CellTiter-Glo 2.0 kit (Promega, SK-N-BE(2)-C, IMR-32, Kelly) or the CellTiter-Glo 3D kit (Promega, NB8) and bioluminescence was read in an OPTIMA plate reader (BMG Labtech).