Polyethylenimine hcl max transfection reagent

Cells were plated on a 0.1% gelatin coated surfaces such that they would be 75% confluent at the time of transfection. Between 30min and 1hr prior to transfection media was changed to DMEM, 10% FBS, 1% NEAA, plus 1% HEPES (Invitrogen), and added at 70% normal volume. For TDP-43 expression experiments, cells were transfected with a total of 2ug DNA per well of a 6-well plate or 1ug DNA per well of a 12-well plate, using a 1:3 ratio of total DNA to PEI (polyethylenimine HCl MAX transfection reagent (Polysciences, Inc)). Media was changed 6hrs after transfection to DMEM, 10% FBS, 1% NEAA. For viruses used in iPSCs, the media post-transfection was switched to mTeSR1 media. In experiments using TDP-43 expression vector, 1ug/ml doxycycline (Hyclate) Hydrochloride (Sigma #9891) was added to media. For aggregation reporter validation and post-screen transfection-based experiments, transfected cells were lifted 24hrs post doxycycline induction and analyzed by FACS (FACSAria Fusion BD).

HEK293T cells were from ATCC (Cat# CRL-3216 RRID:CVCL_0063) and have been authenticated by the vendor and were not contaminated by mycoplasma. HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium, high glucose (Gibco) containing 10% fetal bovine serum (Life Sciences), 1% non-essential amino acids (Gibco), and 1% antibiotic-antimyotic (Gibco). Cells were plated in gelatin-coated 6-well plates at a density of 3 × 10⁶ cells/plate 24 hr before transfection. Cells were transfected with 2 µg total DNA and 7.35 µl polyethylenimine HCl MAX transfection reagent (Polysciences, Inc). Wells co-transfected with mClover3-TDPΔNLS and HSP104 variants received 1 µg of each plasmid. Media was changed 6 hr post-transfection to media containing 1 µg/ml of doxycycline hyclate (Sigma-Aldrich) to induce transgene expression. Transfected cells were lifted every 24 hr over 2 days, at which point cells were analyzed by FACS (FACSAria Fusion BD). Cells were gated to have a narrow range of FCS and SSC values and to be fluorescence positive. TDP-43 aggregation was quantified by comparing the height (FITC-H) to the width (FITC-W) of the fluorescence channel using 488 nm laser and FITC filters.