H4 cells were used as they are of human origin and are derived from a central nervous system tissue (neuroglioma); additionally, they can be readily cultured, have high transfection efficiency, and are suitable for high-content imaging. H4 cells were maintained in Dulbecco's modified Eagle's medium (Sigma–Aldrich, D6546) supplemented with 10% fetal bovine serum (Life Technologies, Inc., 10500064), 2 mm l -glutamine (Sigma–Aldrich, G7513), and 5000 units/ml penicillin/10 μg/ml streptomycin (Sigma–Aldrich, P4458). SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium/F-12 (1:1) (Thermo Fisher Scientific, 21331020) supplemented with 10% fetal bovine serum (Life Technologies, 10500064), 2 mm l -glutamine (Sigma–Aldrich, G7513), and 5000 units/ml penicillin/10 μg/ml streptomycin (Sigma–Aldrich, P4458). All cells were maintained in a humidified incubator at 37 °C, 5% CO2.
H4 and SH-SY5Y cells stably expressing EGFP-PRKN were created using a lentiviral vector. EGFP-PRKN (a kind gift from Dr. Jon Lane, University of Bristol) was cloned into a third-generation lentiviral backbone, and viral particles were produced by co-transfection of HEK293T cells as described previously (58 (link)). Cells were transduced with the virus and, after repeated passaging, sorted by FACS to produce a cell line expressing a consistent level of EGFP-PRKN.
H4 and SH-SY5Y cells stably expressing EGFP-PRKN were created using a lentiviral vector. EGFP-PRKN (a kind gift from Dr. Jon Lane, University of Bristol) was cloned into a third-generation lentiviral backbone, and viral particles were produced by co-transfection of HEK293T cells as described previously (58 (link)). Cells were transduced with the virus and, after repeated passaging, sorted by FACS to produce a cell line expressing a consistent level of EGFP-PRKN.