Mouse hepatic stellate cells (mHSCs) were isolated from C57/Bl6 WT, TLR4−/−, and MyD88−/− mice by enzymatic digestion and Percoll density gradient centrifugation,24 (link) with modifications. Following two to three passages, 50% subconfluent mHSCs were transfected with pCMV Simian Virus 40 Large T antigen22 (link) for 48 hours followed by selection with 100 mg/mL hygromycin B (Invitrogen, San Diego, CA) and grown at 33°C. Immortalized WT, TLR4−/−, and MyD88−/− mHSC lines were generated by single cell clonal expansion and were genotyped as described for the TLR4 knockout mice.25 (link),26 (link)
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Isolation and Immortalization of Mouse Hepatic Stellate Cells
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Cell clonal
Density gradient centrifugation
Digestion
Enzymatic
Hepatic stellate cells
Hygromycin b
Knockout mice
Mouse
Percoll
Simian virus 40
Corresponding Organization :
Other organizations : Fudan University, Shanghai Medical College of Fudan University, Zhongshan Hospital, Icahn School of Medicine at Mount Sinai, Alameda Hospital
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Variable analysis
independent variables
- Genotype of mice (WT, TLR4-/-, MyD88-/-)
- Characteristics of immortalized mouse hepatic stellate cell (mHSC) lines
- Passage number of mHSCs (2-3 passages)
- Subconfluency of mHSCs (50% subconfluent)
- Transfection with pCMV Simian Virus 40 Large T antigen (for 48 hours)
- Selection with hygromycin B (100 mg/mL)
- Growth temperature (33°C)
- Not explicitly mentioned
- Not explicitly mentioned
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