Cellometer auto t4 plus

In order to ensure reproducibility, the cell count was determined prior to each transfection by Cellometer^® Auto T4 Plus (Nexcelom Bioscience, Lawrence, MA, USA). The HEK A10/A17 dKO cells were plated at a density of 3.5 × 10⁵ cells per 6-well. The next day, cells were transfected utilizing polyethylenimine (PEI, 1 µg/µL) in a 1:3 DNA to PEI ratio. After 46 h, the medium was changed and where indicated the cells were stimulated with 200 nM phorbol 12-myristate 13-acetate (PMA) for 2 h and harvested.

In order to ensure reproducibility, the cell count was determined prior to each transfection by Cellometer^® Auto T4 Plus (Nexcelom Bioscience; #SD100). SH-SY5Y CTSD KO cells were seeded into 6-well plates at 2 × 10⁵ cells per well for western blot analysis and in same density onto 12 mm cover glasses for immunofluorescence analyses. After 24 h of expression, cells were transiently transfected with Lipofectamine 2000 (Thermo 611 Fisher Scientific; #11668027) following the manufacturer’s protocol. Cells were washed in PBS and harvested for experimental analysis after 48 h and a-syn co-transfected cells were harvested after 72 h of expression. H4 CTSD KO cells were plated at a density of 4 × 10⁵ per well (6-well plate) and transfected 24 h later using Effectene transfection reagent (Qiagen; #301425) according to the manufacturer’s instructions. H4 CTSD KO cells were harvested after 48 h of expression. For CTSD inhibition, pepstatin A (PepA; Sigma-Aldrich; #508437) was diluted in cell culture media to a final concentration of 100 μM and incubated for 2 days after transfection. Bafilomycin A1 (BafA1) was used to inhibit the lysosome. BafA1 (Santa Cruz; #sc-201550A) was dissolved in DMSO and diluted in cell culture media to a final concentration of 0.2 μM. Cells were treated with BafA1 one day after transfection for 16 h.