Fugene hd in vitro transfection reagent

Reporter constructs were methylated using CpG Methyltransferase M.Sssl (New England Biolabs). Complete methylation was verified by plasmid DNA bisulfite modification and pyrosequencing using specific primers. The immortalized human chondrocytes, C28/I2, were seeded at a density of 30,000 cells per well in 24-well plates, cultured in complete DMEM/F12 + 5% FBS + ITS + A2P + P/S overnight, and transfected with a mixture of 500 ng of luciferase reporter vector and 1 ng of pRL-TK Vector (Promega), using FuGENE HD in vitro Transfection Reagent (Promega) in serum-free conditions. Transfected C28/I2 cells were cultured for an additional period of 48 hours prior to harvest. Cell lysates were assayed for Firefly and Renilla luciferase activities using the Dual-Luciferase Reporter Assay System on a Varioskan Flash Multimode Reader (Thermo Scientific). Firefly luciferase activity of each transfection was normalized against Renilla luciferase activity. The RUNX2 expression vector was used in co-transfection experiments, and the empty pCMV5 backbone served as a control. Reactions were performed in duplicate, and each experiment was repeated 4 times.

Plasmids were methylated using CpG Methylase M.Sssl (New England Biolabs). Complete methylation was verified by plasmid DNA bisulfite modification and pyrosequencing with specific primers. The immortalized human chondrocytes, C28/I2, were seeded at a density of 30,000 cells per well in 24-well plates, cultured in DMEM/F12 overnight, and transfected with a mixture of 300 ng luciferase reporter vector and 1 ng pRL-TK Vector (Promega), using FuGENE HD in vitro Transfection Reagent (Promega). Transfected C28/I2 cells were cultured for 48 h prior to harvest. Cell lysates were assayed for firefly and renilla luciferase activity using a Dual-Luciferase Reporter Assay System on a Varioskan Flash (Thermo Scientific). Firefly luciferase activity of each transfection was normalized against renilla luciferase activity. Reactions were performed in duplicate, and each experiment was repeated at least three times.
The expression vectors for NF-κB (p50, p65, or p50/p65), AP-1(c-Fos, c-Jun, or c-Fos/c-Jun) and C/EBPβ were used (60 ng) for co-transfections. Blank expression vector pCMV4, pcDNA3.1 and pcDNA3.1(+) served as controls, respectively. Total DNA was normalized with empty vectors in the transfection mixture.