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Primary mouse hepatocytes isolation and cell transfection

HM Hoi Yin Mak
QO Qian Ouyang
ST Sergey Tumanov
JX Jiesi Xu
PR Ping Rong
FD Feitong Dong
SL Sin Man Lam
XW Xiaowei Wang
IL Ivan Lukmantara
XD Ximing Du
MG Mingming Gao
AB Andrew J. Brown
XG Xin Gong
GS Guanghou Shui
RS Roland Stocker
XH Xun Huang
SC Shuai Chen
HY Hongyuan Yang
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Primary mouse hepatocytes were isolated from 3-month-old mice using a collagenase-based method and cultured as previously described47. Hepatocytes from preparations with a cell viability ≥90% were seeded in six-well plates at a density of 1 × 106 per well in a plating medium (M199 with GlutaMAXTM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, 0.1% bovine serum albumin (BSA), 10% (v/v) foetal bovine serum, 10 nM insulin, 200 nM triiodothyronine, 500 nM dexamethasone) for 4 h and then incubated overnight (16 h) in a recovery medium (M199 with GlutaMAXTM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10% (v/v) foetal bovine serum). The next morning, cells were used for transfection by plasmids with Lipofectamine-3000 (Thermo Fisher Scientific).

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