Cell transfection

LL Lixiang Liu
JW Jian Wu
YG Yi Gao
YL Yang Lv
JX Jiajia Xue
LQ Lihong Qin
CX Cheng Xiao
ZH Zhongchang Hu
LZ Lichun Zhang
XL Xiaotong Luo
YW Yanli Wang
YC Yang Cao
YC Yang Cao
GZ Guoliang Zhang
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Si-RNA (20 uM) with the best interference efficiency and Si-NC (20 uM) were transfected into preadipocytes. When the degree of the cell fusion reached 75%, the complete medium was replaced with an antibiotic-free medium (petri dish of 35 mm). 5 μl of LIP2000 and Si-RNA (including Si-NC) were respectively diluted with 250 μl serum-free medium. The LIP2000 dilution was placed at the room temperature for 5 minutes, and the Si-RNA (including Si-NC) diluent was placed at the room temperature for 20 minutes, the two were then mixed and added to the antibiotic-free medium. The cells were placed in the incubator (37°C, 5% CO2) and changed to a complete medium that contained 10 mg/mL insulin, 0.5 mM IBMX (Sigma) and 1.0 mM DEX (Sigma) after 12 hours. The cells were incubated for 48 hours (2 days) and replaced with a complete medium containing 10 mg/mL insulin, which was changed after 96 hours (4 days). After that, the complete medium was changed every 48 hours until the 8th day of differentiation.

The Over-Acot2 and Over-NC plasmids were transfected into preadipocytes. When the degree of the cell fusion reached 75%, the complete medium was replaced with an antibiotic-free medium (petri dish of 35 mm). The 2 μg plasmid and 5 μl FuGENE® HD (Promega) were mixed into 100 μl serum-free medium and placed at the room temperature for 15 minutes, the mixture was then dripped into the antibiotic-free medium. The cells were placed in the incubator (37°C, 5% CO2) and changed to a complete medium that contained 10 mg/mL insulin, 0.5 mM IBMX (Sigma) and 1.0 mM DEX (Sigma) after 12 hours. The cells were incubated for 48 hours (2 days) and replaced with a complete medium containing 10 mg/mL insulin, which was changed after 96 hours (4 days). After that, the complete medium was changed every 48 hours until the 8th day of differentiation.

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