Ultra low attachment 96 well microplates

HeLa, a human cervical cancer cell line, and CAL-27, a human head and neck cancer cell line, were purchased from the American Type Culture Centre (ATCC, Manassas, VA, USA) and have catalogue numbers CCL-2 and CRL-2095 respectively. Both cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin and streptomycin (Gibco), and 4 mM GlutaMax (Gibco). For cell dissociation, 0.2% trypysin-EDTA (Gibco) was used.
For 2D and 3D cell models, all cells were initially split from a monolayer of cells at approximately 80% confluency. For the 2D monolayer cell model, cells were plated such that final confluency at the end point is approximately 70% in either six-well plates or 96-well plates, with initial cell count dependent on time and plate type. Once plated, cells are left in the incubator 37 °C and 5% CO₂ for 24 h to ensure adherence, after which experiments are initiated.
For 3D spheroid cell models, cells are plated in ultra low attachment 96-well microplates (Corning, Corning, NY, USA), with 3125 cells for HeLa per well and 12,500 CAL-27 cells per well. The cells are then centrifuged at 350× g for 5 min and left in the incubator at 37 °C and 5% CO₂. Experiments are initiated once the spheroids form, after incubation for approximately 72 h.