E0127

Primary VSMCs were obtained from 6 to 8-week-old C57BL/6 male mouse aortas using collagenase-elastase digestion as follows: aortas were excised, washed in phosphatebuffered saline, and incubated in DMEM (Invitrogen) containing 1 mg/mL of Collagenase type II (LS004176, Worthington Biochemical Corp) for 10 to 15 min. Then, under microscopic guidance, the vessels were stripped of adventitia, minced with scissors and digested with Collagenase type I (C-0130, Sigma) 0.5 mg/mL and Elastase type III (E−0127, Sigma) 0.125 mg/mL in serum-free DMEM with antibiotics at 37°C until most cells were in suspension. The cell suspension was recovered and centrifuged at 400 g for 5 min, then resuspended in DMEM with 20% fetal bovine serum (FBS, HyClone), 2% penicillin–streptomycin, and cultured in plates. The cells used in the experiments were from passages 3 to 5.

The epithelium containing the taste papillae was peeled using an enzymatic–mechanical dissociation protocol slightly modified from those previously reported (Béhé et al. 1990 (link); Bigiani, 2001 (link); Cherkashin et al. 2016 (link)). Once removed, the tongue was placed in a Sylgard Petri dish filled with a standard mammalian Ringer solution containing (in mm): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 Hepes, 10 glucose, pH 7.4, with NaOH. Using an insulin syringe, 0.5 ml Ringer solution containing 2 mg ml⁻¹ dispase II (D4693, Sigma, St Louis, MO, USA), 0.2 mg ml⁻¹ elastase (E0127, Sigma) and 0.7 mg ml⁻¹ collagenase B (C9891, Sigma) was injected under the epithelium. The syringe needle was carefully inserted from the posterior to the most anterior part of the tongue. Injection of the enzymatic solution was performed while the needle was gently withdrawn to allow the solution to reach the entire epithelium. After 30 min of incubation, the epithelium was peeled off from the underlying muscle and the vallate papilla and taste cells were isolated as described in the following sections.