Mammalian cell culture media Human Epidermal Keratinocytes: HEK

Cells are sourced from various tissues to grow them in in-vitro conditions. Therefore, cell specific nutrients are important for their survival, maintenance and growth. Determining the appropriate cell culture media is a challenge if you are growing a cell line or a microorganism for the first time. Established cell lines, primary cells, stem cells, bacteria and Yeast all require varied nutrients from basic to complex. Based on the cell type, one can easy find what media and nutrients your peers have used before you try to reinvent the wheel.

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Protocol tips
Normal human epidermal keratinocytes were isolated from neonatal skin. Frozen keratinocytes were thawed and cultivated at clonal density on a feeder layer of irradiated or mitomycin C–treated 3T3-J2 cells, at 37°C and 10% CO2 in a 3:1 mixture of the DMEM and Ham’s F12 medium supplemented with 10% FCS, 5 µg/ml insulin, 0.4 µg/ml hydrocortisone, 10−10 M cholera toxin, and 2 × 10−9 M triiodothyronine. Human epidermal keratinocytes were also cultivated with CnT-PR medium (CELLnTEC), EpiLife medium containing supplement S7 (Life Technologies), and MCDB153 medium containing bovine pituitary extract (Hashimoto et al., 1994). Keratinocytes were used between passages 2 and 7. The medium was changed every 4 d.
Protocol tips
Third passage Neonatal Human Epidermal Keratinocytes (NHEKs Lonza) were initially cultured in serum-free conditions in Lonza's KGM-Gold media (basal medium supplemented with bovine pituitary extract, human epidermal growth factor, bovine insulin, hydrocortisone, Gentamicin, Amphotericin-B, Epinephrine and Transferrin) in collagen coated (1∶100; Type 1 rat tail collagen (Sigma) : PBS) T25 flasks. The cells were cultured in a 37°C incubator with 5% CO2, with the media being changed every 2 days until approximately 80% confluent.
Protocol tips
Frozen keratinocytes were thawed and cultivated at clonal density on a feeder layer of irradiated or mitomycin C–treated 3T3-J2 cells, at 37°C and 10% CO2 in a 3:1 mixture of the DMEM and Ham’s F12 medium supplemented with 10% FCS, 5 µg/ml insulin, 0.4 µg/ml hydrocortisone, 10−10 M cholera toxin, and 2 × 10−9 M triiodothyronine
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