Gibco s opti mem reduced serum medium

The day before transfection, HeLa cells were seeded into 96-well plates, containing 50 μl folate-free RPMI 1640, at a density of 1.0 × 10⁴ cells per well and incubated for 24 h. Two plasmids, pGL3 (firefly luciferase, 200 ng) and pRLSV40 (Renilla luciferase, 50 ng), were co-transfected using 1 μl Lipofectamine 2000™ (Invitrogen) following the manufacturer's protocol. Plates were incubated for 4 hours at 37°C in a humidified atmosphere with 5% CO₂ after which the medium was removed from each well. Cells were washed twice with 1× phosphate-buffered saline (PBS) after which 50 μl folate-free RPMI 1640 medium (without antibiotics) was added to each well. Each siRNA was diluted in 50 μl 1× Gibco's Opti-MEM Reduced Serum Medium (Invitrogen) on ice and the diluted samples were immediately transferred to the respective wells of the 96-well plate. Plates were gently rocked back and forth for a few minutes and then incubated for an additional 16 h prior to cell lysis.

The day before transfection, HT-29 cells were seeded into 96-well plates, containing 50 μl folate-free RPMI 1640, at a density of 5.0 × 10⁴ cells per well and incubated for 24 h. For plasmid transfection, pGL3 (firefly luciferase, 600 ng) and pRLSV40 (Renilla luciferase, 150 ng) were combined and diluted in 1× Gibco's Opti-MEM Reduced Serum Medium (Invitrogen) to a final volume of 50 μl. This was followed by the addition of 4 μl Lipofectamine^® LTX (Thermo Fisher). After a 30-minute incubation period at room temperature, complexes were transferred to each well and plates were incubated for 6 h at 37°C in a humidified atmosphere with 5% CO₂ after which the medium was removed from each well. Cells were washed twice with 1× phosphate-buffered saline (PBS) after which 50 μl folate-free RPMI 1640 medium (without antibiotics) was added to each well. Each siRNA was diluted in 50 μl 1× Gibco's Opti-MEM Reduced Serum Medium (Invitrogen) on ice and the diluted samples were immediately transferred to the respective wells of the 96-well plate. Plates were gently rocked back and forth for a few minutes and then incubated for an additional 20 h prior to cell lysis.