Phosphate buffered saline 1x

The following reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA): B-27^® supplement 50x, Fluo-4 AM cell permeant, formaldehyde 37%, GlutaMAX™ supplement, human recombinant epidermal growth factor (EGF), MEM non-essential amino acids solution 100x, N-2 supplement 100x, and phosphate buffered saline 1x (PBS). The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): 2,5-hexanedione, γ-Aminobutyric acid (GABA), bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle Medium (DMEM), endosulfan, methylmercury, penicillin-streptomycin solution 100x, Triton™ X-100, and TWEEN^® 20. Matrigel^® was purchased from Corning (Corning, NY, USA). Fetal calf serum (FCS) was purchased from ATCC (Manassas, VA, USA). Human recombinant fibroblast growth factor basic (bFGF) was purchased from Peprotech (Rocky Hill, NJ, USA). Tetrodotoxin (TTX) was purchased from Tocris Bioscience (Bristol, UK).

AT-84 cells were cultured to 80% confluence. Once the cells reached the exponential growth phase, they were washed three times with PBS (Phosphate Buffered-Saline 1X, Thermo Fisher, Villebon sur Yvette, France) and briefly exposed to nonenzymatic cell dissociation buffer (Thermo Fisher) to dislodge the cells. Cells were carefully washed three times with PBS, resuspended in RPMI medium and diluted to the desired concentration. Ten-week-old C3H mice were anesthetized with isoflurane, and a tumor cell suspension (8 × 10⁵ AT84 cells in 30 μL of RPMI medium, or 30 μL of RPMI) were injected into the musculature of the mobile tongue. Buprenorphine was subcutaneously administered to mice (0.1 mg/kg) just after the surgery. Mice were observed daily and treated with buprenorphine if distinctive pain signs were detected. After eight days, mice lost approximately 15–20% of their weight and did not present any pain. At this time, mice had tongue tumors that were approximately a third of the mobile tongue. All experimentations of this study were performed at this stage.