Heracell 150i or 160i 5 co2 incubator

WERI-Rb-1 retinoblastoma cells were maintained in RPMI 1640 Medium (11875135, Gibco) + 10% fetal bovine serum (16140071, Gibco) + 1× Penicillin-Streptomycin (30-002-CI, Corning) at 37°C in a HERAcell 150i or 160i 5% CO₂ incubator (Thermo Fisher Scientific). Cells were passaged every 4 days at approximately 1 × 10⁵ to 2 × 10⁶ cells/mL in uncoated flasks by pelleting at 150 g for 5 min and resuspending in fresh media.

HEK293 cells were passaged 24 h prior to be <60% confluent on the day of transfection. Transfection was done with the Lipofectamine 3000 transfection kit (L3000001, Invitrogen). For a 10 cm dish, 7.5 μl lipofectamine 3000 was mixed with 125 μl Opti-MEM (31985062, Thermo Fisher Scientific) at room temperature (RT) for 5 min, and 5 μl of P3000, 0.5–1 μg DNA (Table 2), and 125 μl Opti-MEM was mixed at RT for 5 min as per manufacturer instruction. The 2 solutions were mixed and incubated at RT for 10 min. Media was aspirated from HEK293 cells and replaced with 8 mL of pre-warmed Opti-MEM. Lipofectamine mixture was added to cells and incubated at 37°C in a HERAcell 150i or 160i 5% CO₂ incubator for 4 to 6 h. Transfection media was aspirated and replaced with HEK293 maintenance media (DMEM/10% FBS/1× Penn/Strep) and cells were allowed to recover overnight. Cells were adhered to slides coated with 10% Poly-L-lysine solution (0.1% w/v in H₂O) (P8920, Sigma) for 1 to 3 h at 37°C in a HERAcell 150i or 160i 5% CO₂ incubator (Thermo Fisher Scientific). Cells were washed in 1× PBS and fixed in 10% neutral buffered formalin (HT501128, Sigma) for 30 min. Slides were either used immediately or ethanol dehydrated and stored at −80°C.

HEK293 cells were maintained in DMEM 4.5 g/L D-glucose, L-glutamine (11965084, Gibco) + 10% fetal bovine serum (16140071, Gibco) + 1× Penicillin-Streptomycin (30-002-CI, Corning) at 37°C in a HERAcell 150i or 160i 5% CO₂ incubator (Thermo Fisher Scientific). Cells were split 1:4 to 1:10 every 2 to 3 days when approximately 80% confluent and kept in 6-well dishes or uncoated flasks using Trypsin (0.25%)-EDTA (0.02%) in HBSS (118-093-721, Quality Biological).