Falcon filter

Mice hippocampus were dissected out from P0-P2 pups and digested by 2.5% Trypsin (GIBCO, Thermo Fisher Scientific) for 15 min at 37 °C with gentle agitation. Tissues were then washed 3 times with DMEM (supplemented with 5% FBS, 1% Glutamax, 1% sodium pyruvate and 1% Pen/Strep) and triturated with a fire-polished pasteur pipette until the suspension is homogenous and filtered by falcon filter (BD Biosciences). Neurons were seeded (0.2–0.3 × 10⁶ cells/well) in 12-well plate on glass coverslips coated with poly-d-lysine for immunofluorescence or in 6-well plate (0.5–0.6 × 10⁶ cells/well) coated with poly-D-lysine. Next morning, DMEM was replaced by Neurobasal medium (NBM, GIBCO, Thermo Fisher Scientific) supplemented with 2% B27, 1% Glutamax, and 1% Pen/Strep for long-term maintenance. Lipofectamine 2000 (Invitrogen) was used for transient transfection according to manufacturer’s instructions at DIV 8-9, alternatively, neurons were infected with lentiviruses expressing corresponding Syn1 proteins.

We first anesthetized the mice by intraperitoneally injecting 7% chloral hydrate (25 mg/g). Five minutes later, the peritoneal cavity was opened. The inferior vena cava was perfused with EGTA solution (Sigma Aldrich). When the liver color became lighter, we cut the portal vein and perfused the liver using the solution of protease and collagenase (Sigma Aldrich). At the end of the perfusion, a white texture was visible on the liver. After perfusion, the mice were sacrificed, and the organ was cut and transferred to a 6 cm culture dish. After the addition of pre-warmed protease and collagenase in vitro hydrolyzate, we disrupted the liver tissue by forceps. Then, the broken liver tissue was transferred into a 50 ml centrifuge tube and incubated with the 20 ml pre-warmed protease and collagenase in vitro hydrolyzate and 1% DNase (Solarbio) in a 37°C hybridization chamber for 20 min. The liver tissue was then filtered by the 70 μm pore size Falcon filter (BD Biosciences Discovery Labware, Bedford, MA) and centrifuged 20 ~ 30 × g for 4 ~ 5 min at 4°C. The bottom primary hepatocytes were finally obtained.