F2442 500ml

Human embryonic kidney (HEK) 293T cells (referred to as 293T cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (F2442-500ML; Sigma, St. Louis, MO, USA). Chicken embryo fibroblasts (DF-1) were grown in DMEM containing 10% FBS (CCS30010.02; MRC, QLD, Australia). The T7 RNA polymerase stably expressing cell line, BSRT7 (kindly gifted by Jingjing Cao, Shandong University, China), was cultured in DMEM containing 10% FBS (F2442-500ML; Sigma, St. Louis, MO, USA) and 500 μg/ml G418 (A100859; Sangon Biotech, Shanghai, China). Avibirnavirus IBDV strain NB is maintained in our laboratory (27 (link)).

Lentivirus was produced in HEK293T cells [available from the American Type Culture Collection (ATCC)] through a triple transfection with a packaging plasmid Gag-Pol and an envelope plasmid pVSV-g together with a lentiviral transfer vector carrying an aSyn gene variant (or a pooled aSyn library). HEK293T cells were plated at 400,000 cells per well in six-well plates and incubated at 37°C. The next day, 800 ng of Gag-pol, 200 ng of pVSV-g, and 1000 ng of lentiviral transfer vector were mixed with 7.5 μl of TransIT-293 (Mirus) and 92.5 μl of reduced serum medium (Gibco, Opti-MEM) and incubated at RT for 15 min. The cell medium was changed to 1 ml of DMEM (Dulbecco’s modified Eagle’s medium; Gibco, 11965118) with 3% fetal bovine serum (FBS; Sigma-Aldrich, F2442-500ML). The transfection mixture was added dropwise to the wells and incubated at 37°C. After 6 hours, the medium was changed to fresh 1.5 ml of DMEM with 3% FBS. After 48 hours, the medium was collected, stored at 4°C, and replaced with fresh 1.5 ml of DMEM with 3% FBS. The next day, the medium was collected, combined with the previous one, and centrifuged at 1000g for 5 min. Polybrene (Sigma-Aldrich Polybrene Infection TR-1002-G) and Hepes were added to the supernatant to a final concentration of 4 μg/ml and 50 nM, respectively. The mixture was passed through a 22-μm filter, aliquoted, and stored at −80°C.

COX cells were obtained from the International Histocompatibility Working Group, Seattle, WA [(IHW09022) http://www.ihwg.org/hla/index.html]. PGF cells were obtained from the Coriell Biorepository (Cat #GM03107). They are both homozygous for chromosome 6. COX HLA typing is: HLA-A 01:01:01:01, HLA-B 08:01:01, HLA-C 07:01:01, HLA-DRB1 03:01:01:01, HLA-DQB1 02:01, HLA-DPB1 03:01. PGF HLA typing is: HLA-A 03:01:01:01, HLA-B 07:02:01, HLA-C 07:02:01:03, HLA-DRB1 15:01:01:01, HLA-DQB1 06:02:01, HLA-DPB1 04:01. Cells were cultured in RPMI-1640 medium with 15% FBS (Sigma Cat # F2442-500ML).