Co-transfection of HEp-2 cells with plasmid constructs and
siRNAs
HEp-2 cells (ATCC® Number: CCL-23™) were cultivated with 10 % fetal
bovine serum (FBS) (HyClone, U.S.) and 1 % penicillin/streptomycin (PS;
PanEco, Russia) in Dulbecco’s modified Eagle’s medium (DMEM; PanEco,
Russia). The cells were seeded into 24-well plates at a concentration of
105 cells/well in 0.5 mL of media and incubated at
37°C with 5% CO2 for 12-24 h. The cells were
consistently co-transfected with
each of the plasmid constructs (0.25 μg/well) pRdRp-full,
pVAX-N-IRES-LUC and pVAX-LP-IRES-LUC/Lipofectamine 3000 (0.5 μL/well)
complexes and then in 30 minutes with each of the 15 different siRNAs
(0.75 μg/well) with Lipofectamine 3000 (1.5 μL/well). In both cases
complexes with Lipofectamine 3000 were made in serum-free Opti-MEM media
(Gibco) according to the manufacturer’s protocol. After four hours,
media with complexes were replaced with fresh DMEM. Then, after 24 h
cells were harvested, lysed, and assayed for firefly luciferase activity
using the Luciferase Assay System (Promega) according to the
manufacturer’s instructions. Cells transfected with siLuc (described
previously(28)) and siGFP were used as a
positive and negative control, respectively.