L glutamine

Embryonic brains were dissected from 14.5 days post coitum (dpc) and placed in Ca²⁺ and Mg²⁺-free Hank's Balanced Salt Solution (HBSS) to remove the cerebellum and meninges. Isolated brains were transferred to trypsinization solution containing 0.05% trypsin/EDTA (Cellgro) and incubated for 30 min at 37°C with vigorous shaking at 250 rpm. Trypsinization was quenched by adding an equal volume of cell culture medium (DMEM supplemented with 10% fetal bovine serum (FBS), 20 mM L-glutamine, and 1% antibiotics/antimycotics (Cellgro)). After centrifugation at 1,000 rpm for 5 min, the tissue was triturated in neuronal cell culture medium (Neurobasal® medium supplemented with B-27® supplement (Invitrogen), 1× GlutaMax, 0.5 mM L-glutamine, and 1% antibiotics/antimycotics (Cellgro)) by gentle pipetting through a 1000 μl pipette tip. Triturated tissues were strained through a 40 μm nylon mesh. Resulting cells were plated on a tissue culture dish coated with poly-D-lysine (MW 30,000–70,000, Sigma-Aldrich) and laminin (Invitrogen) at 1 × 10⁴ to 1.5 × 10⁵ cells/cm² depending on the experiment, and then cultured in the same medium. One-half of the medium was changed every three days.

VERO cells, HeLa cells, CHO cells, and human foreskin fibroblasts (HFF) were purchased from the American Type Culture Collection. All cells were maintained at 37°C in a 5% CO₂ atmosphere and grown in either Alpha MEM (with Earle’s salts without ribonucleosides, deoxyribonucleosides, and glutamine) or DMEM (with 4.5 g/l glucose and sodium pyruvate without l-glutamine; Corning Cellgro). Unless otherwise stated, media was supplemented with 10% (vol/vol) FBS, 2 mM l-glutamine (Corning Cellgro), and 100 U/ml penicillin plus 100 mg/ml streptomycin (Quality Biological). HeLa cells were transiently transfected using 2 µg plasmid DNA and the Amaxa Nucleofector solution R according to the manufacturer’s instructions using program I-013 (Lonza) or with 0.25 µg plasmid DNA and JetPrime reagent according to the manufacturer’s instructions (Polyplus-transfection SA). To engineer a stable cell line expressing GFP-Rab11A, the GFP-Rab11A plasmid was linearized with ApaL1 and transfected into VERO cells using 2 µg of linearized plasmid, the Amaxa Nucleofector solution R, and program V-01, according to the manufacturer’s instructions. Stable clones were selected with 800 µg/ml G418 sulfate (Corning Cellgro) in Alpha MEM with 20 mM Hepes and cloned in serial dilutions in 96-well plates.