Cell cultures and transfection
HEK293T cell lines grown in DMEM GlutaMAX® medium (GibcoTM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin were maintained at 37°C in a humidity-controlled atmosphere with 5% CO2. The ST-hmGlu5 HEK293T cell line enables inducible expression of the hmGlu5 receptor containing a SNAP-tagged extracellular VFT domain, as previously described . Briefly, the release of the constitutive Tet repression of the ST-hmGlu5 gene is induced by doxycycline (Dox), allowing a reproducible and controlled expression, proportional to Dox concentration and incubation time. Cells were maintained in ST-hmGlu5 transgene selection medium in the presence of 100 μg/ml hygromycin B and 15 μg/ml blasticidin. Dox (1µg/ml) was added 6h (Ca2+experiments) or 10 to 24h (TR-FRET measurement) before experiments. Cell lines transduction with the pWPT-EF1a-GcaMP6s-P2A-Scarlet lentivirus (gift from Vincent Compan) enabled stable expression of GcaMP6s fluorescent Ca2+ sensor. Plasmids transfection was performed on adherent cells the day after the seeding in antibiotic-free culture medium using Lipofectamine 2000TM (Life Technologies) diluted in OptiMEM GlutaMAX® (Life Technologies) or jetPEI® (Polyplus, only for patch clamp experiments), 24 to 48h before experiments.
Primary cultures of hippocampal neurons were prepared from C57BL/6 mice P0-2 pups as previously described . Neurons were seeded onto plastic dishes coated with Poly-L-ornithine (0.03 mg/ml) and laminin (1µg/ml) in Neurobasal, B27, Glutamax, Glutamine, serum and 1% penicillin/streptomycin medium. Neurons were then maintained at 37°C with 5% CO2 and incubated from DIV2 to DIV3 with AraC (1 µM) to inhibit glial cell proliferation. The medium was then progressively replaced by a serum-free medium (BrainPhys, B27, Glutamax).