The OCT4-GFP reporter cell line was created as described previously47 (link). Cells were expanded on mitomycin C-treated MEF (mouse embryonic fibroblast) feeder cells with Knockout DMEM containing 10% KSR (knockout serum replacement), 10% human plasmanate (Talecris Biotherapeutics), 1% NEAA (non-essential amino acids), 1% penicillin/streptomycin, 1% Gluta-MAX, and 55 μM 2-mercaptoethanol47 (link). 30 ng/mL of bFGF (basic fibroblast growth factor, Life Technologies) was added daily into the growth medium and cells were passaged using Accutase (Millipore) at ~80% confluency.
Protocol detail
Maintenance of OCT4-GFP Reporter hESCs
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Accutase
Basic fibroblast growth factor
Cell line
Cells
Embryonic
Essential amino acids
Feeder cells
Fibroblast
Growth medium
Human
Mitomycin c
Mouse
Oct4
Penicillin
Plasmanate
Serum
Streptomycin
Corresponding Organization :
Other organizations : University of California, San Diego, La Jolla Bioengineering Institute
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Variable analysis
independent variables
- Concentration of bFGF (basic fibroblast growth factor) added to the growth medium
- OCT4-GFP reporter expression
- Mitomycin C-treated MEF (mouse embryonic fibroblast) feeder cells
- Knockout DMEM containing 10% KSR (knockout serum replacement), 10% human plasmanate, 1% NEAA (non-essential amino acids), 1% penicillin/streptomycin, 1% Gluta-MAX, and 55 μM 2-mercaptoethanol
- Cell passage using Accutase at ~80% confluency
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