Cell culture dishes

A mouse melanoma cell line (B16-F10) and mouse embryo fibroblasts (MEF) (Cell Bank, Shanghai, China) were cultured continuously at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA)18 (link). Cell culture dishes from MatTek Co. (Ashland, MA, USA) were rinsed three times with phosphate buffered saline (PBS) before use.

cDNA for S6K1 and 4EBP1-GFPSpark constructs were obtained from Sino Biological (China); mCherry-raptor, YFP-mTOR, YFP-PRAS40 and FLAG-mTOR from Addgene (USA). EGFP-mTOR, mDsRed-Rheb, EGFP-Rheb and mDsRed-raptor constructs were cloned previously^{21 (link)}. Miniprep and Maxiprep kits purchased from Qiagen (Germany). Rapamycin, L-leucine, L-serine and Ponceau S stain were purchased from Sigma-Aldrich (USA).
HEK293 cell line was purchased from ATCC^® (USA), tested for mycoplasma contamination, and HEK293F cells were provided by Evotec (UK) Ltd. Cell culture dishes (35 mm × 20 mm) were bought from MatTek (USA).
HEK293 and HEK293T cells were cultured in Minimum Essential Media (MEM) supplemented with 10% Foetal Bovine Serum (FBS), 2 mM L-glutamine and 1% Penicillin-Streptomycin (P/S) for HEK293. HEK293F cells were grown in serum-free media (Gibco/Life Technologies, UK). All culture reagents were from Life Technologies. Cells were incubated at 37 °C with 5% CO₂ humidified air in T75 culture flasks (Thermo Fisher Scientific, UK) or in 50 mL tubes (Corning^®). SF9 cells were cultured in SF 900 III media supplemented with 1% P/S at 26 °C.

Cultures of dissociated sensory neurons from adult rat DRG were prepared as described previously [56 (link)]. Under terminal anesthesia, rats were decapitated, and L4–L6 lumbar DRGs were dissected under aseptic conditions, then incubated with 1 mg/ml of collagenase (Roche Diagnostics, Indianapolis, IN, USA) in 0.6% of glucose in PBS for 1 h 30 min followed by trypsin-EDTA treatment (0.025% W/V; Gibco, Montréal, Québec, Canada) for 30 min at 37 °C. To stop enzymatic digestion, a 10× volume of DMEM high-glucose was added. After triturating the ganglia using heat-polished Pasteur pipettes, the sensory neurons were centrifuged (3 min at 800 rpm) and resuspended in DMEM high glucose with equal volume of HAM’s medium mixture F12 (Wisent inc, St-Bruno, Québec, Canada) supplemented with 10% fetal bovine serum heat inactivated (FBS, Wisent inc, St-Bruno, Québec, Canada), 2% of penicillin and streptavidin (Gibco, Montréal, Québec, Canada), and 50 ng/ml of nerve growth factor (NGF, Sigma-Aldrich, St-Louis, MO, USA). Finally, neuronal cells were plated onto poly-d-lysine-laminin-coated coverslips (Sigma-Aldrich, St-Louis, MO, USA) mounted on cell culture dishes (Mattek Corporation, Ashland, MA, USA). Cells were maintained during 15 h for calcium imaging at 37 ^oC in a water-saturated atmosphere with 5% CO₂.