Cell Culture and Transfection
Human HEK293T cell were purchased from ATCC. STHdh (CH00096 – Q111/Q7) was obtained from Coriell Cell Repositories. The patient iPS-derived striatal neurons Q47 cells were obtained from a Mongolian Huntington’s Disease patient. The generation and authentication of Huntington’s Disease iPS-derived neurons were described previously (29) .The study was approved by The Ethic Community of Institutes of Biomedical Sciences at Fudan University.
Human HEK293T cell, mouse STHdh cells were cultured in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). iPSCs were cultured in essential 8 medium (Gibco) , iPS-derived striatal neurons Q47 cells were grown and maintained in neuron medium (Neuronbasal+N2+B27+BDNF+GDNF+IGF+Vitamin C and antibiotics). All the mammalian cell lines were maintained at 37 °C incubator with 5% CO2, except STHdh cells, which were maintained at 33 °C with 5% CO2.
Cells were plated at a density of 30% and split whenever they reached 90% confluence. Before transfection, cells were seeded in 10cm dishes to achieve 70% confluence on the next day. For transfection, 10μg plasmid DNA and 30μL PEI (polyethylenimine, PEI) was added into 500μL MEM (Gibco). The reaction mixture was mixed by pipette. After an incubation of 30min at RT the solution was added dropwise to the cells.
The protocol for neuron siRNAs transfection was described previously (29). The siRNAs were reversely transfected into iPS-derived neurons with lipofectamine RNAiMAX (Life Technologies, #13778). All transfections were performed according to the manufacturer’s protocol. Cells were collected 3 days after siRNA transfection or 2 days after cDNA transfection for Western-blot, HTRF or Immunofluorescence analysis of neuronal loss and apoptosis. The siRNA target sequences and/or order information:
Scrambled siRNA: non-targeting siRNA: UUCUCCGAACGUGUCACGUTT (Invitrogen, #D-001810-10);
HTT siRNA: targeting 5’-CAGGUUUAUGAACUGACGUUA-3.