Adult skin specimens obtained from Caesarean sections and circumcisions were used to establish the cell cultures. Keratinocyte cultures were obtained by suspending individual epidermal cells derived according to published methods (22 (link)), in EpiLife Medium supplemented with bovine pituitary extract (BPE), bovine insulin (BI), hydrocortisone, human epidermal growth factor, and bovine transferrin (BT) (Invitrogen). Early passages from 3 to 4 were used for these experiments. Melanocyte cultures were obtained by suspending individual epidermal cells in Medium 254 supplemented with BPE, foetal bovine serum (FBS), BI, hydrocortisone, bFGF, BT, heparin, and phorbol 12-myristate 13-acetate (Invitrogen). Fibroblast cultures were obtained by suspending individual dermal cells lacking epidermis (23 ) in DMEM-supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Invitrogen). Cytokeratins 14 and 10 (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA) were used as markers for keratinocytes; c-kit (mouse monoclonal; Santa Cruz Biotechnology) and tyrosinase-related protein 1 (goat polyclonal; Santa Cruz Biotechnology) were used as markers for melanocytes; and ER-TR7 (rat polyclonal; Santa Cruz Biotechnology) as a marker for fibroblasts (24 (link)–26 (link)).
Protocol detail
Isolation and Culture of Skin Cell Types
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EpiLife Medium
hydrocortisone
human epidermal growth factor
Medium 254
foetal bovine serum
bFGF
heparin
phorbol 12-myristate 13-acetate
penicillin
streptomycin
ER-TR7
Adult
Bovine
C kit
Caesarean sections
Cell cultures
Cells
Circumcisions
Cytokeratins 14
Epidermal cells
Epidermal growth factor
Epidermis
Fibroblasts
Foetal bovine serum
Goat
Heparin
Human
Hydrocortisone
Insulin
Keratinocytes
Melanocytes
Mouse
Penicillin
Phorbol myristate acetate
Streptomycin
Transferrin
Tyrosinase related protein 1
Corresponding Organization :
Other organizations : Dongguk University Ilsan Hospital
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Variable analysis
independent variables
- Source of skin specimens (Caesarean sections and circumcisions)
- Establishment of cell cultures (keratinocytes, melanocytes, fibroblasts)
- Expression of cell-specific markers (cytokeratins 14 and 10 for keratinocytes, c-kit and tyrosinase-related protein 1 for melanocytes, ER-TR7 for fibroblasts)
- Early passages from 3 to 4 for the cell culture experiments
- Culture media (EpiLife Medium for keratinocytes, Medium 254 for melanocytes, DMEM with 10% FBS for fibroblasts)
- None specified
- None specified
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