Culturing Salivary Carcinoma Spheres

Non-adherent spheres of salivary mucoepidermoid carcinoma cells (salispheres), previously characterized in normal salivary cells [57 (link)], were cultured in DMEM/F-12 (Invitrogen) supplemented with 20 ng/ml EGF (Sigma-Aldrich), 20 ng/ml basic fibroblast growth factor (bFGF; Millipore), 1% penicillin/streptomycin (Invitrogen), 1% glutamax (Invitrogen), 1% N-2 supplement (Invitrogen), 1 μM dexamethasone (Sigma-Aldrich), and 10 μg/ml insulin (Sigma-Aldrich) [39 (link)]. Cells were counted, diluted to 2,000 per 1.5 ml, and added to 6-well ultra-low attachment plates (Corning; Corning, NY, USA). For in vitro passaging, salispheres were collected and exposed to 0.25% trypsin for 5-10 minutes, and then mechanically dissociated. The trypsin was neutralized using a trypsin neutralizing solution (TNS; Lonza). Colonies of 50 cells or more were considered salispheres.