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).