The Flp-In™ T-REx™ HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM GlutaMAX-I, 10% FBS, and 100 U/mL HyClone penicillin-streptomycin at 37 °C and 5% CO2.
The pcDNA5-GFP-Vimetin construct was generated by inserting vimentin cDNA (NCBI Reference Sequence: NM_003380.5) into the pcDNA5-GFP backbone. Plasmid mCherry-LaminA-C-18 (#55068) was ordered at ADDGENE.
The cells were seeded at 1 to 2 × 105 per 6-well, and plasmid transfections were performed using Lipofectamine™ 3,000 transfection reagent. The fluorescent-positive cells were sorted with FACS into a 6-well plate. The positive cells were grown in a selection medium with G418 (200 μg/mL), hygromycin (100 μg/mL), and blasticidin (15 μg/mL). The expression of the vimentin protein was induced with tetracycline at 1 μg/mL for 24 h.
The pcDNA5-GFP-Vimetin construct was generated by inserting vimentin cDNA (NCBI Reference Sequence: NM_003380.5) into the pcDNA5-GFP backbone. Plasmid mCherry-LaminA-C-18 (#55068) was ordered at ADDGENE.
The cells were seeded at 1 to 2 × 105 per 6-well, and plasmid transfections were performed using Lipofectamine™ 3,000 transfection reagent. The fluorescent-positive cells were sorted with FACS into a 6-well plate. The positive cells were grown in a selection medium with G418 (200 μg/mL), hygromycin (100 μg/mL), and blasticidin (15 μg/mL). The expression of the vimentin protein was induced with tetracycline at 1 μg/mL for 24 h.