Lenti x accelerator

SH‐SY5Y human neuroblastoma cells (Sigma‐Aldrich #94030304) at P11 were transduced in a 6‐well plate using 300 µl of Auto‐hLAG3 LV pre‐incubated with 30 µl of Lenti‐X™ Accelerator (Clontech #631256) for 30 min. The LV mixture was added onto the cells, evenly spread over the whole well and put into incubator onto a neodymium magnetic sheet (supermagnete #NMS‐A4‐STIC; adhesive force of 450 g/cm²) for 10 min. Cells were then removed from the incubator, LV mixture was completely removed, and fresh SH media added. Cells were kept until confluency and then sub‐cultured with the neodymium magnet sheet kept under the plate. To remove remaining magnetic beads, cell pellet was resuspended in 1.5 ml of SH media, pipetted into 1.5‐ml Eppendorf tube and inserted into DynaMag™‐2 Magnet (Invitrogen #12321D). Cell suspension was then removed from the tube leaving all magnetic beads in the tube. Quantification of P11+1 SH‐10 cells showed that 76% of cells were expressing hLAG3 upon induction by 1 µg/ml of Doxycycline (DOX; Clontech #631311). This decreased to 59% in P11+2 and remained around 50% in the following passages. P11+3 was used for all experiments except for the CO‐IP, where a later passage was used.