Polyethylenimine max

HEK293 cells were grown in Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose (GE Healthcare) supplemented with 10% (v/v) fetal bovine serum and 2 mM L-glutamine. Cells were transfected using TurboFect (Thermo Scientific) or with polyethylenimine MAX (Polysciences) according to the manufacturer’s instructions. Cells were analysed 24 h post-transfection.
Rat cortical neurons were obtained from embryonic day 17 embryos, plated on poly-L-lysine coated 18 mm diameter glass coverslips (Marienfield GmbH & Co.KG) or on 35 mm diameter glass bottom μ-dishes (Ibidi GmbH) and cultured in Neurobasal medium containing B27 supplement (Invitrogen), 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin. Neurons were transfected using Lipofectamine 2000 (Invitrogen) (2 μl/μg DNA in Opti-MEM) according to the manufacturer’s instructions and analysed 24 h post transfection on DIV6 or 7. siRNAs were applied at DIV2 for 96 h.

AAV-PHP.eB viruses were packaged in AAVpro 293T cells (Clontech, 632273). Cells were harvested by cell lifter (Biologix, 70-2180) at 72 h after cotransfection with PHP.eB (Addgene, 103005), pAdDeltaF6 (Addgene, 112867) and transfer plasmids using polyethylenimine MAX (Polysciences, 24 765). Cell pellets were suspended in 1× gradient buffer (10 mm Tris-HCl, pH 7.6, 150 mm NaCl, 10 mm MgCl₂). Cells were lysed by five repeated cycles of freezing in liquid nitrogen for 7 min, thawing in 37°C water bath for 3 min, and vortexing for 2 min. Cell lysate was mixed with ≥50 U/ml of Benzonase nuclease (Millipore, E1014) and incubated at 37°C for 30 min. After centrifugation at 20,000 × g for 30 min at 4°C, the supernatant was transferred to a iodixanol (Optiprep, D1556) step gradient (15%, 25%, 40%, and 58%) for ultracentrifugation. After centrifugation at 40,000 rpm for 4 h at 4°C, virus particles were collected from the layer of 40% iodixanol gradient using a sterile syringe. Purified AAVs were concentrated using Amicon filters (EMD, UFC801096) and formulated in sterile PBS supplemented with 0.01% Pluronic F68 (Invitrogen, 24040032). Virus titers were determined by qPCR using a linearized AAV plasmid as a standard.

Cells were seeded into 6-well plates. About 18 to 24 h later, cells were transfected with plasmids using Lipofectamine LTX Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Polyethylenimine Max (Polysciences) was used for plasmid transfection in HEK293T cells. Twenty-four to 48 h after transfection, cells were used for further experiments.
For the isolation of CHO-K1 cells harboring pFLAG-CH25H^tet-on (CHO-CH25H^tet-on cells), CHO-K1 cells seeded into 6-well plate were cotransfected with pFLAG-CH25H^tet-on (2 μg/well) and pMAM-BSD (0.1 μg/well). Twenty-four hours after transfection, cells were grown in medium containing blasticidin (7 μg/ml) for 5 days. Blasticidin-resistant clones were isolated by limited dilution, and clones that were positive for Dox-dependent FLAG-CH25H expression were selected by immunoblotting with anti-FLAG antibody.