S cell line 96 well nucleofector kit sf

LCLs GM18856 and GM19093 were counted (Vi-Cell, Beckman Coulter, Fullerton, CA, USA) and diluted to 5×10⁵ cells/ml 24 hours prior to nucleofection. Cells (viability >85%) were nucleofected using Lonza’s Cell Line 96-well Nucleofector Kit SF (Lonza Inc, Basel, Switzerland). The reaction was performed according to the manufacturer’s protocol using 1×10⁶ cells per well and 2000 nM final concentration of AllStars Negative Control siRNA (Qiagen, Valencia, CA, USA) or a pool of IKZF3 FlexiTube siRNA (Hs_IKZF3_1, Hs_IKZF3_2, Hs_IKZF3_3 and Hs_ZNFN1A3_5)(Qiagen, Valencia, CA, USA). The cells were nucleofected using the DN-100 program on an Amaxa Nucleofector 96-well Shuttle (Lonza Inc, Basel, Switzerland). After a 10 minute rest, 85uL pre-warmed media was added to each transfected well and cells were allowed to rest for an additional 5 minutes. Cells were plated for mRNA pelleting in 24-well flat bottom plates. Cells were harvested at 5, 29 and 53 hours after nucleofection, washed in ice-cold PBS and centrifuged to remove PBS. All pellets were stored at −80°C until RNA isolation.