Matrigel coated coverslips

Primary hippocampal neuronal cultures were prepared from newborn mice of either sex of C57BL/6 J (JAX: 000664) or conditional knockout mouse line that is homozygous for Mint1 and 3 knockout and floxed for Mint2 (Mint1^−/−; fMint2//fMint2; Mint3^−/−). All animal experiments were approved by Boston University Institutional Animal Care and Use Committee. All methods were performed in accordance with relevant guidelines and regulations. Neurons were dissociated with trypsin 10 min at 37 °C, triturated and plated onto matrigel-coated coverslips (BD Bioscience). Neurons were maintained in a humidified incubator with 5% CO₂ at 37 °C. Recombinant lentiviruses were generated by transfecting HEK293T cells with lentiviral plasmids for Δ (a control vector without cre recombinase), Cre, Mint2 WT, Mint2 N723S, or Mint2 ΔPDZ with viral enzymes and envelope proteins (pRSV/REV, pMDLg/RRE, and pVSVG) using FuGENE6 reagent. The initial media was changed into neuronal growth media after 8 h of transfection. Lentivirus-containing conditioned media was collected after 48 h, centrifuged at 500 g for 10 min at 4 °C to remove cell debris and stored at −80 °C.

Neurospheres were prepared as previously described^{37 (link)}. Briefly, E15.5 embryos were individually dissected in PBS and the neocortex was transferred in Dulbecco’s Modified Eagle Medium/F12 (DMEM) and dissociated by extensive enzymatic digestion with Papaine (Sigma-Aldrich). Cells were grown in medium containing DMEM/F12, 0.66% glucose, L-glutamine 1%, Pen/Strep 1%, 4 µg/mL of heparin, hormone mix with the addition of either 20 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast growth factor (bFGF). In such conditions, cells spontaneously formed neurospheres. Neurospheres were then dissociated into single-cell cultures and plated on matrigel-coated coverslips (BD Bioscience).