3d cell titerglo assay

Cell viability of 2D-cultured cells was determined using a Z2 Coulter Counter Analyzer (Beckman) or by the CyQuant cell proliferation assay (Invitrogen). Cell viability of 3D-cultured organoids was determined by the 3D Cell TiterGlo assay (Promega) on a GloMax plate reader (Promega).

The cells (5 × 10³ cells/mL) were seeded into a 96-well, clear, round-bottom, ultra-low attachment Microplate (Corning Costar, Cambridge, MA, USA) for 7 days to form spheroid cells [15 (link)], and sphere viability was measured by the 3D CellTiter Glo assay (Promega, Madison, WI, USA) [18 (link)].

Cells were seeded, either as a mono-culture or co-culture using a 1:1 ratio (500 CAFs: 500 cancer) in Ultra-Low Attachment (ULA) plates at 1000 cells/well and incubated for 24 h to form spheroids. The cell number and drug incubation were determined in PANC-1 cells using a cell density gradient assay over a number of time-points (48, 72 and 96 h) to determine the most optimal assay conditions. The cells were treated with gemcitabine or paclitaxel and incubated for 72 h. The cell viability of 3D spheroids of pancreatic cancer cell lines and/or co-cultures was determined using the Promega 3D CellTiter-Glo assay. Briefly, the CellTiter-Glo 3D reagent was thawed at 4 °C overnight and then placed in a 22 °C water bath for 30 min before use. The plate containing the spheroids was equilibrated for 30 min at room temperature. Fifty microlitres of CellTiter-Glo 3D was added to each well and placed on an orbital plate shaker for 5 min. The cell lysate and CellTiter-Glo reagent were moved to a flat, clear bottom 96-well plate, incubated for a further 25 min (room temperature), and then the luminescent signal was measured using an EnVision multilabel plate reader (Perkin Elmer).